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
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ABSTRACT |
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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 g1 · 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
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INTRODUCTION |
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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.
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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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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.
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RESULTS |
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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 g1 · 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 g1 · 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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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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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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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 g1 · 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-2 IR
and thyroid hormone receptor-
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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50255 (H. Yang) 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.
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