Hypothyroidism induces Fos-like immunoreactivity in ventral medullary neurons that synthesize TRH

Pu-Qing Yuan and Hong Yang

CURE: Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, and Department of Medicine, Division of Digestive Diseases and Brain Research Institute, University of California Los Angeles, Los Angeles, California 90073


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Altered thyroid statuses are associated with autonomic disorders. Thyrotropin-releasing hormone (TRH) in medullary nuclei regulates vagal efferent activity. Induction of Fos-like immunoreactivity (IR) in medullary TRH-synthesizing neurons was investigated in 24-h fasted rats with different thyroid statuses. Hypo- and hyperthyroidism were induced by 6-N-propyl-2-thiouracil (PTU) in drinking water and a daily intraperitoneal injection of thyroxine (T4; 10 µg · 100 g-1 · day-1), respectively, for 1-4 wk. The numbers of Fos-like IR positive neurons in the raphe pallidus, raphe obscurus, and parapyramidal regions, which were low in euthyroid rats (0-2/section), increased remarkably as the hypothyroidism progressed and were negatively correlated with serum T4 levels. At the 4th wk, Fos-like IR positive neurons were 10- to 70-fold higher compared with euthyroid controls. Simultaneous T4 replacement (2 µg · 100 g-1 · day-1) prevented the increases of Fos-like IR in PTU-treated rats. Hyperthyroidism did not change the number of Fos-like IR neurons in the raphe nuclei but reduced it in the parapyramidal regions. Double immunostaining revealed that most of the Fos-like IR induced by hypothyroidism was located in the prepro-TRH IR positive neurons. The selective and sustained induction of Fos-like IR in TRH-synthesizing neurons in ventral medullary nuclei by hypothyroidism indicates that these neurons play a role in the autonomic disorders observed in altered thyroid statuses.

thyroid hormones; thyroxine; raphe pallidus; raphe obscurus; parapyramidal regions


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ABNORMAL THYROID STATUSES are associated with changes in visceral functions (46, 47). Hypothyroidism induces sinus bradycardia and increases gastric acid secretion and ulcer formation (8, 46, 47), whereas hyperthyroidism induces tachycardia and decreases gastric acid secretion (21, 46, 47). The peripheral mechanisms responsible for these disorders, especially for those in the heart, have been extensively studied (54). However, the exact nature of interactions between thyroid status and autonomic nervous activities in the control of visceral organs, particularly the central mechanisms, is still poorly understood. Although thyroid hormone has profound effects in the central nervous system, its central regulation of autonomic function has received little attention.

Growing evidence obtained in recent years has revealed that specific nuclei located in the ventral regions of the medulla, namely the raphe pallidus (Rpa), raphe obscurus (Rob), and parapyramidal regions (PPR), play important roles in the central regulation of peripheral autonomic activities (52). These nuclei contain neurons with similar neurochemistry and projections (31, 52). Neuronal terminals arising directly from these nuclei innervate the dorsal motor nucleus of the vagus (DMN) (31) and the intermediolateral cell column of the spinal cord (44). TRH, substance P, and serotonin are among the neurotransmitters synthesized by these nuclei and released from the neuronal terminals to modulate the function of preganglionic motoneurons of the vagus and sympathetic nervous systems (4, 31, 44, 52). It is well established that TRH in medullary nuclei plays a physiological role in vagal regulation of gastric function by increasing vagal efferent discharges (37, 52). The caudal raphe nuclei (Rpa and Rob) and the PPR contain the most abundant groups of TRH-synthesizing neurons outside of the hypothalamus (28, 58). Dense TRH-containing nerve terminals and TRH receptors are located in the dorsal vagal complex (DVC) and the nucleus ambiguus (32, 41), which are the main medullary sources of vagal nerves regulating the heart and gastrointestinal tract (29). Exogenous injection of TRH into the DMN, or induction of endogenous TRH release into the DMN by chemical stimulation of the neurons in the Rpa (13, 23, 55, 57), Rob (50), or PPR (61), induced bradycardia (57) and increased gastric acid secretion (55, 61), motility (13, 50), and ulcer formation (23). The bradycardia and gastrointestinal changes induced by chemical stimulation of the raphe nuclei could be prevented by pretreatment both with TRH antibody microinjected into the DVC (55) or nucleus ambiguus (57) and with antisense oligodeoxynucleotides of TRH receptor injected intracisternally (50). Taken together, these findings provide strong evidence that TRH-containing caudal raphe/PPR projections to the vagal motoneurons play an important role in the brain stem regulation of the peripheral autonomic nervous system.

Thyroid hormones exert a feedback regulation on TRH gene expression in the paraventricular nucleus (PVN) of the hypothalamus (26, 48). Decreased thyroid hormone levels caused by thyroidectomy induce Fos expression in the TRH-synthesizing neurons in the PVN (24). Fos is the product of c-fos gene and is a member of the set of cellular inducible transcription factors (ITFs) (17, 35). These factors are induced by diverse extracellular stimuli and interact with DNA to influence the transcription of specific genes, including the TRH gene (42, 49). The expressions of Fos and other ITFs in the central nervous system are therefore widely used as markers of neuronal activation by specific stimuli (11, 15, 18, 36). In particular, Fos may be involved in the feedback regulation of TRH gene expression by thyroid hormones (24).

To investigate the central mechanisms through which hypo- or hyperthyroidism influences autonomic activity, we hypothesized that the autonomic disorders observed in altered thyroid statuses may be related to the influence of thyroid hormones on TRH-synthesizing neurons in the medullary Rpa, Rob, and PPR. We have recently reported that thyroid hormones exert a feedback regulation on TRH mRNA levels in these nuclei (59). In the present study, the expression of Fos-like protein, as observed by immunohistochemistry, was used as a marker to evaluate activity-dependent alterations of the neuronal function in these nuclei in different thyroid statuses. Double immunostaining was used to observe whether Fos-like immunoreactivity (IR) was localized specifically in the TRH-synthesizing neurons. The correlations between the numbers of Fos-like IR positive neurons in these ventral medullary nuclei and serum T4 levels were studied in hypothyroid animals.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Animals and treatments. Male Sprague-Dawley rats weighing 270-320 g (Harlan, CA) were maintained on rat Purina chow and tap water ad libitum and housed under conditions of controlled temperature (22 ± 2°C) and illumination (light on 0600-1800) for at least 7 days before any treatment. Rats were then divided into four groups and treated for 4 wk: 1) euthyroid, injected intraperitoneally daily with vehicle (0.02 N NaOH/saline); 2) hypothyroid, induced by 0.1% 6-N-propyl-2-thiouracil (PTU; Sigma, St. Louis, MO) added to the drinking water and a daily intraperitoneal injection of vehicle; 3) hypothyroid (0.1% PTU in the drinking water) with T4 (Sigma) replacement (daily ip injection of T4, 2 µg/100 g); and 4) hyperthyroid, induced by a daily intraperitoneal injection of a higher dose of T4 (10 µg/100 g). In the time-course study, four groups of rats drinking 0.1% PTU without daily intraperitoneal injections were killed after 1, 2, 3, or 4 wk of treatment. The corresponding control groups received no treatment. At the end of treatments, all rats were fasted for 24 h and then killed by transcardial perfusion under deep pentobarbital (70 mg/kg ip, Abbott Laboratories, North Chicago, IL) anesthesia. Blood samples (0.5 ml/rat) were collected from the left ventricle before the perfusion, and sera were kept at -75°C before measurement of total T4 levels. Brain stems were collected for immunohistochemistry of Fos-like protein and prepro-TRH. All animal protocols were approved by the Veterans Administration Medical Center/West Los Angeles Research Service Animal Committee.

T4 RIA. Serum aliquots (10 µl) were used to measure total T4 levels with a commercial RIA kit (ICN Biomedicals, Costa Mesa, CA). The sensitivity of the assay ranged from 0 to 25 µl/dl. All samples were measured in duplicate.

Fos-like immunohistochemistry and quantitative analysis. Fos-like IR was detected as previously described (5). Rats fasted for 24 h were deeply anesthetized with pentobarbital and transcardially perfused with 100 ml of isotonic saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were removed, postfixed for 3 h at 4°C in the same fixative, and subsequently cryoprotected overnight in 20% sucrose in 0.1 M PB. Coronal frozen sections (30 µm) of the brain stem were cryostat cut (Microtome, IEC, MA) at the interaural levels of -1.80 to -5.08 mm according to the atlas of Paxinos and Watson (38). This included the whole rostral-caudal length of the Rob, PPR, and most of the Rpa in the ventral medulla. Free-floating sections were incubated for 24 h at 4°C with the primary antibody (Fos Ab-5 rabbit polyclonal antibody, Oncogene Research Products; dilution 1:10,000 in 0.01 M PBS containing 0.3% Triton X-100 and 3% normal goat serum) followed by 1 h at room temperature with a biotinylated secondary antibody (goat anti-rabbit, Jackson ImmunoResearch Laboratories, West Grove, PA; dilution 1:1,000). Sections were finally processed for avidin-biotin-peroxidase with the use of diaminobenzidine as the chromogen and then were mounted on slides (Superfrost/Plus, Fisher Scientific, Pittsburgh, PA), dehydrated in ethanol, cleared in xylene, and coverslipped. The presence of Fos-like IR was detected with bright-field microscopy as a dark brown reaction product in the cell nuclei.

Because the Rpa and the Rob sizes vary from the rostral to caudal levels (38), the number of neurons in each of these nuclei at different levels is also varied. To make data comparable, we divided both the Rpa and the Rob into three regions respectively: rostral (r), interaural, -1.80 to -2.60 mm; middle (m), -2.60 to -4.30 mm; and caudal (c), -4.30 to -5.08 mm (Fig. 1). The regions designated rRpa and mRob contain the greatest density of neurons within the Rpa and Rob, respectively (Fig. 1). The levels used for PPR were from -1.80 to -2.80 mm, which contains most PPR neurons (Fig. 1). The numbers of Fos-like IR positive neurons in different regions of the Rpa, Rob, and PPR were counted and quantified as the average number from 20 sections per region in each rat. Both left and right PPRs were added together and taken as one region. Double-counting errors were corrected by the following formula proposed by Abercrombie (2) to estimate nuclear populations from microtome sections: P = A × [M/(L + M)], with P being the corrected cell count, A the total cell count, M the section thickness (µm), and L the average diameter of the nucleus (µm). To determine the average diameter of the nucleus, 10 randomly selected Fos-like IR positive nuclei were measured with a microruler in each section and at least five sections per region were measured.


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Fig. 1.   Coronal brain stem sections showing location and size of raphe pallidus (Rpa), raphe obscurus (Rob), and parapyramidal regions (PPR) in rostral, middle, and caudal medulla. Regions were defined as: caudal, interaural -4.30 to -5.08 mm; middle, -2.60 to -4.30 mm; and rostral, -1.80 to -2.60 mm. Sections are adapted from atlas of Paxinos and Watson (38).

Double immunostaining for Fos-like IR and prepro-TRH IR. After incubations with Fos antibody and biotinylated goat anti-rabbit IgG as described in Fos-like immunohistochemistry and quantitative analysis, sections were processed for avidin-biotin-peroxidase with the use of diaminobenzidine enhanced with nickel ammonia sulfate as the first chromogen. Sections were then rinsed with PBS for 3 h at room temperature and incubated with a rabbit polyclonal antibody raised against prepro-TRH160-169 (1128B6, diluted 1:2,000, gift from Dr. Eugene Pekary) in PBS containing 0.1% Triton and 3% normal goat serum. Sections were finally processed by an avidin-biotin-peroxidase procedure with diaminobenzidine as the second chromogen. Fos-like IR was detected as a dark blue reaction product in the nuclei, and prepro-TRH IR appeared as a brown reaction product in the cytoplasm. Inactivation of the antibody (1128B6) by incubation with prepro-TRH160-169 (PS4, Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr) completely abolished the immunostaining for this peptide.

Statistical analysis. Quantitative data are expressed as the means ± SE of each group. Comparisons between two groups were analyzed by Student's t-test, and multiple groups were compared by two-way ANOVA with a statistical program (SigmaStat 2.03). Correlations between serum T4 levels and the numbers of Fos-like IR positive neurons in specific brain stem regions were analyzed by linear correlation. P values less than 0.05 were considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Serum T4 levels in different thyroid statuses after 4 wk of treatment. Serum T4 levels in euthyroid rats were 3.1 ± 0.4 µg/dl. Rats drinking 0.1% PTU had 84% lower serum T4 levels (0.5 ± 0.0 µg/dl) compared with euthyroid rats. Daily injection of T4 (2 µg · 100 g-1 · day-1) in PTU-treated rats prevented this decrease and brought serum T4 levels to 2.6-fold higher than the euthyroid controls (8.0 ± 0.5 µg/dl). Animals with hyperthyroidism induced by daily intraperitoneal injection of a high dose of T4 (10 µg · 100 g-1 · day-1) showed a sixfold higher level of serum T4 (19.2 ± 1.7 µg/dl) compared with the euthyroid rats.

Numbers of Fos-like IR positive neurons in the Rpa, Rob, and PPR in different thyroid statuses. Rats injected intraperitoneally with vehicle for 4 wk had only a few Fos-like IR positive neurons in each of the observed regions in the Rpa, Rob, and PPR (Table 1; Figs. 2-4). Hypothyroidism selectively induced remarkable increases of Fos-like IR positive neurons in the Rpa, Rob, and PPR by 10- to 70-fold but not in the surrounding areas or in other nuclei within the caudal ventral medulla (Table 1; Figs 2-4). The most abundant numbers of Fos-like IR positive neurons were observed in the rRpa compared with other observed regions (Table 1; Fig. 2). Simultaneous T4 replacement (2 µg · 100 g-1 · day-1) in PTU-treated rats significantly, although not completely, prevented the induction of Fos-like IR in the Rpa, Rob, and PPR by 62-95% (Table 1; Figs. 2-4). Daily high dose of T4 injection (10 µg · 100 g-1 · day-1) did not significantly change the number of Fos-like IR positive neurons in the raphe nuclei (Table 1; Figs. 2 and 3) but significantly decreased Fos-like IR in the PPR compared with euthyroid controls (Table 1).

                              
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Table 1.   Number of Fos-like IR positive neurons in Rpa, Rob, and PPR in different thyroid statuses after 4 wk of treatments



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Fig. 2.   Tendency of the number of Fos-like immunoreactivity (IR) positive neurons in Rpa and Rob in different thyroid statuses after 4 wk of treatment. Each column is mean of 20 sections · region-1 · rat-1 and 4-6 rats/group. Control, euthyroid; 6-N-propyl-2-thiouracil (PTU), hypothyroid rat induced by 0.1% PTU in drinking water; PTU + thyroxine (T4), hypothyroid + T4 replacement (2 µg · 100 g-1 · day-1 ip); T4, hyperthyroid rat induced by T4 injection (10 µg · 100 g-1 · day-1 ip). Rostral, interaural, -1.80 to -2.60 mm; middle, -2.60 to -4.30 mm; caudal, -4.30 to -5.08 mm according to atlas of Paxinos and Watson (38).



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Fig. 3.   Light view micrographs of rostral (r) Rpa and middle (m) Rob showing Fos-like IR positive neurons in different thyroid statuses after 4 wk of treatment. Control, euthyroid; PTU, hypothyroid rat induced by 0.1% PTU in drinking water; PTU + T4, hypothyroid + T4 replacement (2 µg · 100 g-1 · day-1 ip); T4, hyperthyroid rat induced by T4 injection (10 µg · 100 g-1 · day-1 ip). Arrows in control sections indicate location of nuclei. rRpa, coronal sections at level of interaural -2.60 mm (50×); mRob, coronal sections at level of interaural -3.80 mm (85×) according to atlas of Paxinos and Watson (38).



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Fig. 4.   Light view micrographs of PPR showing Fos-like IR positive neurons in different thyroid statuses. Control, euthyroid; PTU, hypothyroid rat induced by 0.1% PTU in drinking water; PTU + T4, hypothyroid rat with T4 replacement (2 µg · 100 g-1 · day-1 ip); T4, hyperthyroid rat induced by T4 injection (10 µg · 100 g-1 · day-1 ip). Arrows in control section indicate location of PPR. Sections are at level of interaural -3.72 mm (150×) according to atlas of Paxinos and Watson (38).

Negative correlations between serum T4 levels and the numbers of Fos-like IR positive neurons in the Rpa, Rob, and PPR. Correlations between serum T4 levels and the numbers of Fos-like IR positive neurons in the Rpa, Rob, and PPR were observed during the development of hypothyroidism. Serum T4 levels gradually decreased as the PTU treatment continued, and they reached a significantly low level of 16% compared with the control value after 2 wk of treatment. Thereafter, T4 levels remained low until the end of the 4-wk treatment period (Fig. 5A). During progression of the hypothyroidism, there were significant negative correlations between serum T4 levels and the numbers of Fos-like IR positive neurons in all regions examined (i.e., the r, m, and c regions of the Rpa, Rob, and PPR) (Table 2; Fig. 5). The negative correlation between T4 levels and the numbers of Fos-like IR in the rRpa are shown in Fig. 5 as an example in graph form.


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Fig. 5.   Time courses of serum T4 levels (A) and number of Fos-like IR positive neurons (B) in rRpa during progress of hypothyroidism. Columns are means ± SE of 4-6 rats as indicated in Table 2. * P < 0.05 compared with control levels. C: correlation between serum T4 levels and number of Fos-like IR positive neurons in rRpa. r, Correlation coefficient.


                              
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Table 2.   Time courses of increase of Fos-like IR positive neurons in Rpa, Rob, and PPR during progress of hypothyroidism and their correlations with serum T4 levels

Colocalization of Fos-like IR and prepro-TRH IR in the Rpa, Rob, and PPR in hypothyroid rats. The immunostaining of prepro-TRH IR with antiserum 1128B6 in colchicine-treated euthyroid rats showed positive neurons clearly confined within the Rpa, Rob, and PPR of the medulla (data not shown). Prepro-TRH IR could not be detected in the medulla in euthyroid rats without colchicine pretreatment. However, because colchicine itself induces Fos-like IR in many brain areas including the Rpa, Rob, and PPR (data not shown), double-staining analyses were not performed in euthyroid rats. In hypothyroid rats, prepro-TRH IR could be observed in these nuclei without colchicine pretreatment. Double immunostaining for prepro-TRH IR and Fos-like IR in hypothyroid rats without colchicine treatment showed that over 90% of the Fos-like IR positive neurons in the Rpa, Rob, and the PPR were also prepro-TRH IR positive (Fig. 6).


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Fig. 6.   Neurons double stained with Fos-like IR and prepro-TRH IR in mRpa, rRpa, mRob, and PPR (250×). Dark blue-stained nuclei indicate presence of Fos-like IR, and light brown staining in cytoplasm indicates presence of prepro-TRH IR.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The present findings demonstrate that hypothyroidism induced by PTU results in remarkable induction of Fos-like IR within ventral medullary TRH-synthesizing neurons. In 24-h fasted euthyroid rats, only a few Fos-like IR positive neurons were observed in the medulla; that is consistent with previous observations (5). In contrast, PTU added to the drinking water gradually decreased serum thyroid hormone levels and increased the number of Fos-like IR positive neurons in the Rpa, Rob, and PPR by 10- to 70-fold. The induction of Fos-like IR in these nuclei does not result from postprandial events, which have been known to induce Fos-like IR in the brain stem nuclei regulating vagal activity (11). We assessed Fos-like IR in animals fasted for 24 h to avoid the possible induction of Fos expression in the central nervous system by food intake. A 24-h fasting empties the stomach in rats fed with Purina chow and does not induce Fos expression in the Rpa, Rob, and PPR as observed in previous studies (5) and in the present study in euthyroid rats. However, because fasting may cause some changes in metabolism, the possibility of its influence on Fos induction in hypothyroid rats could not be completely excluded. Also, the increase of Fos-like IR observed in hypothyroid rats does not correlate with circadian variations or environment temperature (5), because rats in all groups were kept under similar illumination and temperature controls. Data obtained in the present study indicate that the increases in the numbers of Fos-like IR positive neurons in the Rpa, Rob, and PPR in PTU-treated rats mainly result from the reduction in circulating thyroid hormone levels. This was supported by the time-course study showing negative correlations between the numbers of Fos-like IR positive neurons in these nuclei and serum T4 levels. In addition, T4 replacement inhibited the PTU-induced Fos expression by 62-95%. The increases in Fos-like IR in the Rpa, Rob, and PPR in PTU-treated rats were clearly confined to these nuclei and not in the surrounding areas, indicating that the induction is highly nucleus selective. In addition, the time course of the induction of ventral medullary Fos-like IR in PTU-treated rats, which became significant after 1 wk of treatment, was similar to the onset of Fos expression in the PVN after thyroidectomy, which appeared at 6 days after the surgery (24). Taken together, these findings suggest that in addition to the neurons in the PVN, neurons in the medullary caudal raphe nuclei and the PPR are responsive to decreased levels of circulating thyroid hormone.

The T4 replacement dose selected in the present study (2 µg/100 g) was based on previous reports (48). However, it brought serum T4 to higher levels than euthyroid controls. Previous studies have documented that thyroid hormone-dependent reversal of hypothyroidism-induced changes, such as the rise in hypothalamic TRH gene expressions (22, 27), changes in heart rate (14), and nuclear thyroid hormone receptor levels in the anterior pituitary (27), required high doses of thyroid hormone administration, which induced supraphysiological and hyperthyroid circulating levels. This phenomenon is consistent with the result obtained from the present study showing that when the serum T4 levels in PTU-treated rats with T4 replacement were 2.6-fold higher than the euthyroid T4 levels, the inhibition of the induction of Fos-like IR in the ventral medullary nuclei by PTU was still incomplete.

Hyperthyroidism induced by high doses of T4 injection (10 µg · 100 g-1 · day-1) did not influence the number of Fos-like IR positive neurons in the caudal raphe nuclei but slightly and significantly decreased the Fos-like IR in the PPR compared with euthyroid rats. It is unknown whether neurons in the PPR are more sensitive to hyperthyroidism than neurons in the raphe nuclei. Because euthyroid rats displayed very low basal levels of Fos-like IR positive neurons in both the raphe nuclei and the PPR (<2/section), it was difficult to show a further reduction, even though it may be induced by hyperthyroidism, with such a background level. However, evidence supports that hyperthyroidism may influence activity of neurons in these nuclei. For instance, in humans with a hyperthyroid state, the cardiac vagal motoneurons are less excitable (25). Recently, we found that hyperthyroidism decreased TRH mRNA levels in the caudal raphe nuclei and the PPR by in situ hybridization (unpublished data), although the decrease had been undetectable by Northern blot analysis (59). Future studies should use a more sensitive parameter to evaluate the neuronal activities in these nuclei or assess the effect of hyperthyroidism under a stimulated background, such as cold stress, which induces Fos expression in these nuclei (5).

The Rpa and the PPR are located in the ventral surface of the caudal medulla (38). These anatomic locations enable their neurons to be closely in contact with the cerebrospinal fluid (CSF). Thyroid hormones are mainly transported from blood to the brain across the blood-brain barrier (10). Transthyretin, the major high-affinity thyroid hormone-binding protein in rat plasma, is actively and independently synthesized in the choroid plexus (10). Free T4 and triiodothyronine (T3) concentrations in the CSF are 2.4 and 5.6 times higher, respectively, than those in the serum (16). Also, total T4 levels in the CSF parallel circulating T4 levels in euthyroid, hypothyroid, or hyperthyroid statuses (16). Although the detailed mechanisms through which thyroid hormone induces Fos expression in the neurons in the Rpa, Rob, and PPR are still to be investigated, thyroid hormone receptor isoforms have been recently identified in these nuclei by immunohistochemistry (60). In addition, thyroid hormone receptor-beta 2 IR and thyroid hormone receptor-alpha 1 IR colocalized with prepro-TRH IR by double staining (60, 62). These findings suggest a direct action of thyroid hormone on these neurons.

In vitro studies have provided evidence that c-fos expression may be regulated by thyroid hormone. The nuclear thyroid hormone receptor represses transcription activation by the transcription factor activator protein-1 (AP-1) in a thyroid hormone-dependent fashion (45). Thyroid hormone receptors suppress c-fos by binding to their response elements in its promoter and acting as transcriptional silencers (63). T3 decreased c-fos mRNA levels and the mRNA response to other stimuli and reduced the abundance of nuclear proteins that bind to an AP-1 binding site and the levels of c-Fos protein (40). T3 also strongly decreased basal and stimuli-induced c-fos promoter activity (40). Our result, that Fos-like IR was induced in the ventral medullary nuclei by reduced circulating thyroid hormone levels, is consistent with these in vitro findings. On the other hand, the increase of Fos-like IR may not present an effect of hypothyroidism per se. Indirect mechanisms, such as mediations by other neurotransmitters and/or peptides that innervate the caudal raphe/PPR neurons and the possibility that their actions were influenced by thyroid status, cannot be excluded. Hypothyroid-induced metabolic disorder, hypothermia, and other complications may also mainly or partly contribute to the Fos induction in the raphe and the PPR, because acute cold exposure has been reported to induce Fos expression in these nuclei (5).

TRH, substance P, and serotonin are among the neuropeptides and transmitters that are located in the caudal raphe nuclei and the PPR (31, 44). We chose to examine whether hypothyroidism induces Fos-like IR in TRH-synthesizing neurons in these medullary nuclei because hypothyroidism has been shown to increase prepro-TRH mRNA levels (26, 48) and induce Fos expression in TRH-synthesizing neurons in the PVN of the hypothalamus (24). The tripeptide TRH is difficult to fix with immunohistochemical procedures. Immunohistochemistry with antisera against cryptic portions of prepro-TRH, which are relatively easier to fix and still retain high IR, has been used instead to localize the TRH-synthesizing neurons in the brain (28). The antiserum used in the present study, 1128B6, is specific to the NH2 terminus of PS4 (39). The specific and highly confined immunostainings in the Rpa, Rob, and PPR revealed by this antiserum in colchicine-treated rats (data not shown) confirm previous findings that PS4 was located in TRH-synthesizing neurons in the medullary raphe nuclei (7) and functioned as a TRH-enhancing factor in the DMN (56). In euthyroid rats, prepro-TRH IR in these nuclei could not be immunostained without colchicine pretreatment due to low intracellular levels. Interestingly, prepro-TRH IR could be detected in these nuclei in PTU-treated rats without colchicine treatment, indicating an increase of intracellular prepro-TRH protein contents in these neurons in hypothyroid rats. Although the increased prepro-TRH content may be the result of delays in intracellular transport or the metabolism of the peptides due to the hypothyroid state, it is most likely to be the result of increased synthesis of the peptide. This is supported by the results from a previous study showing that pro-TRH mRNA levels in these nuclei increased in hypothyroid rats (59). Increased TRH synthesis in the caudal raphe and the PPR is also coincident with the autonomic disorders observed in hypothyroidism (46, 47, 52), which are similar to the effects of activation of the medullary raphe/PPR-DMN TRH system (52). The increased prepro-TRH contents in hypothyroid rats facilitated the double-immunostaining study by avoiding colchicine pretreatment, which causes additional Fos expression (24).

Our results show that over 90% of the Fos-like IR positive neurons in the Rpa, Rob, and PPR were also prepro-TRH IR positive. It is known that Fos interacts with a product of another immediate early gene, jun, forming a heterodimeric transcription factor that binds specifically to the AP-1 site and regulates the expression of late response genes (49), including the TRH gene (42). Thyroidectomy inducted Fos (24) as well as TRH gene expressions in the PVN (26, 48). In cultured hypothalamic neurons, TRH gene was coexpressed with both c-fos and c-jun in the same neurons, and the three mRNAs increased in response to glucocorticoid (30). These findings indicate that c-Fos could mediate the effects of hypothyroidism and glucocorticoids on regulating TRH gene expression. Similar to the observations in the PVN of the hypothalamus (26, 48), prepro-TRH mRNAs in the caudal raphe nuclei and the PPR were significantly elevated after surgical thyroidectomy, and the elevations were prevented by T4 replacement (59). The unanimous changes of prepro-TRH mRNA levels (59) and Fos-like IR in these ventral medullary nuclei in different thyroid statuses, together with the colocalization of Fos-like IR with prepro-TRH IR, indicate that Fos-like ITF may be involved in the increased TRH synthesis in medullary caudal raphe/PPR nuclei induced by hypothyroidism.

Fos expression is generally considered transient after the introduction of a stimulus (19), whereas in the present study, the increase of Fos-like IR in the raphe nuclei and the PPR not only was sustained over 4 wk but actually increased progressively during that period. This prolonged existence of Fos-like IR raises the question as to whether the IR tested in the present study is really c-Fos or is actually other Fos-related antigens (FRAs), which have relatively longer half-life (17, 19). There are several considerations regarding this question. First, the antibody specificity is critical. Antibodies generated against the portion of c-Fos that includes the leucine zipper that is essential for the formation of dimers are most likely to detect c-Fos and FRAs equally well (19). On the other hand, antibodies generated against the NH2 terminus of c-Fos are most effective for localizing c-Fos with little cross-reactivity with FRAs (19). The antibody used in the present study (Oncogene Sciences, Ab-5) was produced against the NH2 terminus of the c-Fos protein (Fos4-17) and is therefore considered highly specific (19). In addition, low and restricted expression of c-fos and its protein in the adult nervous system is a common finding (17), whereas FRAs usually present a strong baseline in many brain regions in unstimulated animals (19). The low-level expression of the tested IR in the present study in euthyroid rats indicates that the IR is more likely to be Fos rather than other FRAs. Second, because the same gene products might have different functions in different cells, the regulation of Fos expression as well as the Fos regulation of target gene expression must be determined individually for a specific system (19). There are reports showing that Fos expressions were increased in central nervous system after chronic stimuli, such as social stress (33), dehydration (34), hypoglycemia (6), intermittent hypoxia (15), arthritis (1), and nicotine administration (36). Also, c-fos mRNA levels were persistently elevated in specific brain cells in newborn rats after the mother was exposed to nicotine on gestational days 4-21 (51). The mechanisms for the long-term induction of Fos expression are various among specific cases and may involve distinct signaling pathways (6) but are almost certainly due to processes that continue or are initiated during the stimulation or even after the stimulation per se has ceased (17). It is worth noting that hypothyroidism was not a dull or repeated stimulation in the present study. The serum thyroid hormone levels were consistently changing (declining) during the treatment period. In the PVN, Fos IR increased at 6 days but not at 1 and 3 days after thyroidectomy (there were no data available on longer term observation) (24). We also observed a gradual increase of Fos-like IR positive neurons in the ventral medullary nuclei in the present study, in correlation with circulating T4 levels. These findings indicate that the induction of Fos in specific nuclei by hypothyroidism may be influenced by the threshold of individual neurons in response to the changes of thyroid hormone levels, whereby the total number of Fos positive neurons in a specific nucleus is thyroid hormone concentration dependent. Finally, considering the complex interactions between the ITFs (17) and the complex metabolism alterations induced by hypothyroidism, the induction of ITFs other than Fos in the ventral medullary TRH neurons by hypothyroidism cannot be excluded. A recent study (9) revealed that some FRAs, such as Delta Fos B protein, exhibit a remarkably long half-life. The present finding that hypothyroidism induces Fos-like IR in specific ventral medullary nuclei may not exactly answer how many and which ITFs are involved in the action; however, it does provide a reliable background for further investigations on the interactions between the ITFs and the role of ITFs in the TRH gene expression in these nuclei induced by hypothyroidism.

TRH-containing projections from the Rpa, Rob, and PPR to vagal preganglionic motoneurons in the DMN and the nucleus ambiguus are important medullary pathways regulating vagal efferent activities to the heart and the gastrointestinal tract (20, 32, 37, 41, 50, 52, 53, 55, 58). Fos-like IR can be induced in these nuclei by stimuli influencing autonomic nerve activity, such as stimulation of the carotid sinus nerve (12) and cold stress (5). Cold stress stimulates medullary TRH gene expression and release (58), as well as increases in gastric acid secretion and motility, and induces ulcer formation through activating vagal efferent fibers (3, 58). Because hypothyroidism is associated with significant alterations in autonomic function (46, 47), the selective induction of Fos-like IR in the TRH-synthesizing neurons in the Rpa, Rob, and PPR by hypothyroidism provides important information for understanding the central mechanism through which thyroid hormones regulate autonomic activities. Other mechanisms may also contribute to the autonomic disorders of thyroid diseases, such as thyroid hormone action in the central noradrenergic system (43).

In summary, hypothyroidism induced Fos-like IR in the neurons of medullary Rpa, Rob, and PPR. The Fos-like IR induction was highly selective to these nuclei, negatively correlated with serum T4 levels, prevented by T4 replacement, and mainly localized in TRH-synthesizing neurons. Although the mechanism needs to be further studied, these data, together with the well-established medullary TRH pathway regulating autonomic outflow (20, 32, 37, 41, 50, 52, 53, 55, 58), clearly indicate that the functional change of TRH-synthesizing neurons in the Rpa, Rob, and PPR is one of the mechanisms by which hypothyroidism alters autonomic nervous system function.


    ACKNOWLEDGEMENTS

We thank Dr. Eugene Pekary (Department of Medicine, University of California Los Angeles and Endocrine Section, Veterans Affairs Medical Center/West Los Angeles) for providing the prepro-TRH antiserum and Paul Kirsch for assistance in the preparation of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50255 (H. Yang) and DK-41301 (CURE Animal Core).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. Yang, CURE:DDRC, West Los Angeles VA Medical Center, Bldg 115, Rm. 203, 11301 Wilshire Blvd, Los Angeles, CA 90073 (E-mail: hoyang{at}ucla.edu).

Received 28 December 1998; accepted in final form 10 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbadie, C., and J. M. Besson. c-Fos expression in rat lumbar spinal cord during the development of adjuvant-induced arthritis. Neuroscience 48: 985-993, 1992[Medline].

2.   Abercrombie, M. Estimation of nuclear population from microtome sections. Anat. Rec. 94: 239-247, 1946.

3.   Arai, I., M. Muramatsu, and H. Aihara. Body temperature dependency of gastric regional blood flow, acid secretion and ulcer formation in restraint and water-immersion stressed rats. Jpn. J. Pharmacol. 40: 501-504, 1986[Medline].

4.   Backman, S. B., and J. L. Henry. Effect of substance P and thyrotropin-releasing hormone on sympathetic preganglionic neurones in the upper thoracic intermediolateral nucleus of the cat. Can. J. Physiol. Pharmacol. 62: 248-251, 1984[Medline].

5.   Bonaz, B., and Y. Taché. Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion. Brain Res. 652: 56-64, 1994[Medline].

6.   Brown, E. R., and P. E. Sawchenko. Hypophysiotropic CRF neurons display a sustained immediate-early gene response to chronic stress but not to adrenalectomy. J. Neuroendocrinol. 9: 307-316, 1997[Medline].

7.   Bulant, M., A. Delfour, H. Vaudry, and P. Nicolas. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates pro-TRH-connecting peptides. Identification and characterization of prepro-TRH-(160-169) and prepro-TRH-(178-199) in the rat nervous system. J. Biol. Chem. 263: 17189-17196, 1988[Abstract/Free Full Text].

8.   Chang, H. C., and J. H. Sloan. Influence of experimental hypothyroidism upon gastric secretion. Am. J. Physiol. 80: 732-734, 1927.

9.   Chen, J., M. B. Kelz, B. T. Hope, Y. Nakabeppu, and E. J. Nestler. Chronic Fos-related antigens: stable variants of deltaFosB induced in brain by chronic treatments. J. Neurosci. 17: 4933-4941, 1997[Abstract/Free Full Text].

10.   Dratman, M. B., F. L. Crutchfield, and M. B. Schoenhoff. Transport of iodothyronines from bloodstream to brain: contributions by blood: brain and choroid plexus: cerebrospinal fluid barriers. Brain Res. 554: 229-236, 1991[Medline].

11.   Emond, M. H., and H. P. Weingarten. Fos-like immunoreactivity in vagal and hypoglossal nuclei in different feeding states: a quantitative study. Physiol. Behav. 58: 459-465, 1995[Medline].

12.   Erickson, J. T., and D. E. Millhorn. Fos-like protein is induced in neurons of the medulla oblongata after stimulation of the carotid sinus nerve in awake and anesthetized rats. Brain Res. 567: 11-24, 1991[Medline].

13.   Garrick, T., M. Prince, H. Yang, G. Ohning, and Y. Taché. Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats. Brain Res. 636: 343-347, 1994[Medline].

14.   Goldman, M., M. B. Dratman, F. L. Crutchfield, A. S. Jennings, J. A. Maruniak, and R. Gibbons. Intrathecal triiodothyronine administration causes greater heart rate stimulation in hypothyroid rats than intravenously delivered hormone. Evidence for a central nervous system site of thyroid hormone action. J. Clin. Invest. 76: 1622-1625, 1985[Medline].

15.   Greenberg, H. E., A. L. Sica, S. M. Scharf, and D. A. Ruggiero. Expression of c-fos in the rat brainstem after chronic intermittent hypoxia. Brain Res. 816: 638-645, 1999[Medline].

16.   Hagen, G. A., and W. J. Elliott. Transport of thyroid hormones in serum and cerebrospinal fluid. J. Clin. Endocrinol. Metab. 37: 415-422, 1973[Medline].

17.   Herdegen, T., and J. D. Leah. Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res. Rev. 28: 370-490, 1998[Medline].

18.   Hoffman, G. E., W. S. Lee, M. S. Smith, R. Abbud, M. M. Roberts, A. G. Robinson, and J. G. Verbalis. c-Fos and Fos-related antigens as markers for neuronal activity: perspectives from neuroendocrine systems. NIDA Res. Monogr. 125: 117-133, 1993[Medline].

19.   Hoffman, G. E., M. S. Smith, and J. G. Verbalis. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front. Neuroendocrinol. 14: 173-213, 1993[Medline].

20.   Ishikawa, T., H. Yang, and Y. Taché. Medullary sites of action of the TRH analogue, RX 77368, for stimulation of gastric acid secretion in the rat. Gastroenterology 95: 1470-1476, 1988[Medline].

21.   Johansson, H., and G. Nylander. Effects of thyroxine and thiouracil treatment on gastric secretion and gastric ulcer incidence in the Shay rat. Acta Chir. Scand. 127: 527-535, 1964.

22.   Kakucska, I., W. Rand, and R. M. Lechan. Thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is dependent upon feedback regulation by both triiodothyronine and thyroxine. Endocrinology 130: 2845-2850, 1992[Abstract].

23.   Kaneko, H., and Y. Taché. TRH in the dorsal motor nucleus of vagus is involved in gastric erosion induced by excitation of raphe pallidus in rats. Brain Res. 699: 97-102, 1995[Medline].

24.   Koibuchi, N., R. B. Gibbs, M. Suzuki, and D. W. Pfaff. Thyroidectomy induces Fos-like immunoreactivity within thyrotropin-releasing hormone-expressing neurons located in the paraventricular nucleus of the adult rat hypothalamus. Endocrinology 129: 3208-3216, 1991[Abstract].

25.   Kollai, B., and M. Kollai. Reduced cardiac vagal excitability in hyperthyroidism. Brain Res. Bull. 20: 785-790, 1988[Medline].

26.   Koller, K. J., R. S. Wolff, M. K. Warden, and R. T. Zoeller. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus. Proc. Natl. Acad. Sci. USA 84: 7329-7333, 1987[Abstract].

27.   Lechan, R. M., and I. Kakucska. Feedback regulation of thyrotropin-releasing hormone gene expression by thyroid hormone in the hypothalamic paraventricular nucleus. Ciba Found. Symp. 168: 144-158, 1992[Medline].

28.   Lechan, R. M., and T. P. Segerson. Pro-TRH gene expression and precursor peptides in rat brain. Observations by hybridization analysis and immunocytochemistry. Ann. NY Acad. Sci. 553: 29-59, 1989[Medline].

29.   Loewy, A. D., and K. M. Spyer. Vagal preganglionic neurons. In: Central Regulation of Autonomic Function, edited by A. D. Loewy, and K. M. Spyer. London: Oxford University Press, 1990, p. 68-87.

30.   Luo, L. G., and I. M. Jackson. Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons. Brain Res. 791: 56-62, 1998[Medline].

31.   Lynn, R. B., M. S. Kreider, and R. R. Miselis. Thyrotropin-releasing hormone-immunoreactive projections to the dorsal motor nucleus and the nucleus of the solitary tract of the rat. J. Comp. Neurol. 311: 271-288, 1991[Medline].

32.   Manaker, S., and G. Rizio. Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J. Comp. Neurol. 290: 516-526, 1989[Medline].

33.   Matsuda, S., H. Peng, H. Yoshimura, T. C. Wen, T. Fukuda, and M. Sakanaka. Persistent c-fos expression in the brains of mice with chronic social stress. Neurosci. Res. 26: 157-170, 1996[Medline].

34.   Miyata, S., W. Matsunaga, H. Mondoh, T. Nakashima, and T. Kiyohara. Effect of AV3V lesions on Fos expression and cell size increases in magnocellular neurons of the rat hypothalamus during chronic dehydration. Neurosci. Res. 26: 149-156, 1996[Medline].

35.   Morgan, J. I., and T. Curran. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci. 14: 421-451, 1991[Medline].

36.   Nisell, M., G. G. Nomikos, K. Chergui, P. Grillner, and T. H. Svensson. Chronic nicotine enhances basal and nicotine-induced Fos immunoreactivity preferentially in the medial prefrontal cortex of the rat. Neuropsychopharmacology 17: 151-161, 1997[Medline].

37.   O-Lee, T. J., J. Y. Wei, and Y. Taché. Intracisternal TRH and RX 77368 potently activate gastric vagal efferent discharge in rats. Peptides 18: 213-219, 1997[Medline].

38.   Paxinos, G., and C. Watson. The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1997, p. 1-60.

39.   Pekary, A. E., A. Sattin, and R. L. Lloyd. Electroconvulsive seizures increase levels of PS4, the TRH-enhancing peptide [prepro-TRH(160-169)], in rat brain. Neuroendocrinolog y65: 377-384, 1997[Medline].

40.   Perez, P., A. Schonthal, and A. Aranda. Repression of c-fos gene expression by thyroid hormone and retinoic acid receptors. J. Biol. Chem. 268: 23538-23543, 1993[Abstract/Free Full Text].

41.   Rinaman, L., R. R. Miselis, and M. S. Kreider. Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci. Lett. 104: 7-12, 1989[Medline].

42.   Rondeel, J. M., and I. M. Jackson. Molecular biology of the regulation of hypothalamic hormones. J. Endocrinol. Invest. 16: 219-246, 1993[Medline].

43.   Rozanov, C. B., and M. B. Dratman. Immunohistochemical mapping of brain triiodothyronine reveals prominent localization in central noradrenergic systems. Neuroscience 74: 897-915, 1996[Medline].

44.   Sasek, C. A., M. W. Wessendorf, and C. J. Helke. Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neuroscience 35: 105-119, 1990[Medline].

45.   Schmidt, E. D., S. J. Cramer, and R. Offringa. The thyroid hormone receptor interferes with transcriptional activation via the AP-1 complex. Biochem. Biophys. Res. Commun. 192: 151-160, 1993[Medline].

46.   Seely, E. W., and G. H. Williams. Gastrointestinal manifestations of endocrine disease. In: Principles and Practice of Endocrinology and Metabolism, edited by K. L. Becker. Philadelphia, PA: Lippincott, 1990, p. 1503-1506.

47.   Seely, E. W., and G. H. Williams. The cardiovascular system and endocrine disease. In: Principles and Practice of Endocrinology and Metabolism, edited by K. L. Becker. Philadelphia, PA: JB Lippincott, 1990, p. 1496-1501.

48.   Segerson, T. P., J. Kauer, H. C. Wolfe, H. Mobtaker, P. Wu, I. M. Jackson, and R. M. Lechan. Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus. Science 238: 78-80, 1987[Medline].

49.   Sheng, M., and M. E. Greenberg. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4: 477-485, 1990[Medline].

50.   Sivarao, D. V., Z. K. Krowicki, T. P. Abrahams, and P. J. Hornby. Intracisternal antisense oligonucleotides to TRH receptor abolish TRH-evoked gastric motor excitation. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1372-G1381, 1997[Abstract/Free Full Text].

51.   Slotkin, T. A., E. C. McCook, and F. J. Seidler. Cryptic brain cell injury caused by fetal nicotine exposure is associated with persistent elevations of c-fos protooncogene expression. Brain Res. 750: 180-188, 1997[Medline].

52.   Taché, Y., and H. Yang. Role of medullary TRH in the vagal regulation of gastric function. In: Innervation of the Gut: Pathophysiological Implications, edited by D. L. Wingate, and T. F. Butkd. Boca Raton, FL: CRC, 1994, p. 67-80.

53.   Taché, Y., H. Yang, and H. Kaneko. Caudal raphe-dorsal vagal complex peptidergic projections: role in gastric vagal control. Peptides 16: 431-435, 1995[Medline].

54.   Wickenden, A. D., R. Kaprielian, T. G. Parker, O. T. Jones, and P. H. Backx. Effects of development and thyroid hormone on K+ currents and K+ channel gene expression in rat ventricle. J. Physiol. (Lond.) 504: 271-286, 1997[Abstract].

55.   Yang, H., G. V. Ohning, and Y. Taché. TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G880-G886, 1993[Abstract/Free Full Text].

56.   Yang, H., and Y. Taché. Prepro-TRH-(160-169) potentiates gastric acid secretion stimulated by TRH microinjected into the dorsal motor nucleus of the vagus. Neurosci. Lett. 174: 43-46, 1994[Medline].

57.   Yang, H., and Y. Taché. Thyroid hormone modulates TRH gene expression in caudal raphe nuclei: implication of autonomic regulation. In: Program of the Falk Symposium #77: Gastrointestinal Tract and Endocrine System. Freiburg, Germany: Kluwer Academic, 1994, p. 9-9.

58.   Yang, H., S. V. Wu, T. Ishikawa, and Y. Taché. Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei: relationship with vagally mediated gastric erosions. Neuroscience 61: 655-663, 1994[Medline].

59.   Yang, H., P. Yuan, V. Wu, and Y. Taché. Feedback regulation of thyrotropin-releasing hormone gene expression by thyroid hormone in the caudal raphe nuclei in rats. Endocrinology 140: 43-49, 1999[Abstract/Free Full Text].

60.   Yang, H., P. Q. Yuan, and Y. Taché. Direct action of thyroid hormone on medullary TRH gene expressionin caudal raphe neurons: implication in autonomic regulation (Abstract). Neurogastroenterol. Motil. 10: 353, 1998.

61.   Yang, H., P. Q. Yuan, and Y. Taché. Functional evidence that the medullary parapyramidal regions participate in the medullary regulation of gastric function (Abstract). Gastroenterology 114: A1193, 1998.

62.   Yuan, P. Q., Y. Taché, and H. Yang. Thyroid hormone receptor a1 mRNA in TRH immunoreactive neurons in the rat caudal raphe nuclei (Abstract). Soc. Neurosci. Abstr. 23: 430, 1997.

63.   Zhang, X. K., K. N. Wills, M. Husmann, T. Hermann, and M. Pfahl. Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities. Mol. Cell. Biol. 11: 6016-6025, 1991[Medline].


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