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. Author manuscript; available in PMC: 2021 Apr 29.
Published in final edited form as: Endocrinology. 1999 Jan;140(1):43–49. doi: 10.1210/endo.140.1.6422

Feedback Regulation of Thyrotropin-Releasing Hormone Gene Expression by Thyroid Hormone in the Caudal Raphe Nuclei in Rats

HONG YANG 1, PUQING YUAN 1, VINCENT WU 1, YVETTE TACHÉ 1
PMCID: PMC8083015  NIHMSID: NIHMS1695523  PMID: 9886805

Abstract

Medullary TRH regulates autonomic activity, and altered thyroid status is associated with autonomic disorders. We investigated whether thyroid hormone exerts a negative feedback regulation on TRH gene expression in the medullary caudal raphe nuclei. Medullary pro-TRH messenger RNAs (mRNAs) were mainly located in the raphe pallidus and raphe obscurus neurons as shown by in situ hybridization and were significantly increased by 70% and 160–230% by Northern blot analyses in 24 h fasted rats at 1 and 3–5 weeks after thyroidectomy, respectively, when serum T4 levels were reduced by 75–87%. The increased pro-TRH mRNA on the 30th day after thyroidectomy was reversed to euthyroid levels by T4 replacement (2 or 4 μg/100 g·day). T4 injections (10 or 100 μg/100 g·day for 30 days) did not significantly influence medullary pro-TRH mRNA levels in sham-operated rats. Thyroidectomized rats fed normally showed a 500% increase in pro-TRH mRNA levels 30 days after the surgery, while those fasted for 24 h showed only a 180% increase. These data indicate that medullary TRH gene expression is enhanced during hypothyroidism due to the lack of negative feedback regulation by thyroid hormone, and this response is modulated by feeding state. These findings may have important relevance to understanding autonomic-related visceral alterations induced by hypothyroidism.

HYPO- AND HYPERTHYROIDISM are associated with autonomic nervous system-related disorders, characterized by significant changes in cardiovascular and gastrointestinal function (1, 2). Hypothyroidism induces sinus bradycardia and increases gastric acid secretion and ulcer formation (1–4), whereas hyperthyroidism has opposite effects (1, 2, 5, 6). All attempts to show a direct relationship between the known calorigenic effects of thyroid hormones and the visceral changes have been unsuccessful (7, 8), and the underlying mechanisms involved in the functional alterations are still poorly understood. It is worth noting that the disorders mentioned above could be ascribed to abnormal parasympathetic and sympathetic activities. In addition, thyroid hormones alleviate bradycardia in hypothyroid animals more rapidly and efficiently when injected into the cerebrospinal fluid than iv (9). Therefore, it is possible that thyroid hormone impacts directly on brain nuclei that regulate autonomic nervous system outflow.

Thyroid hormones have profound effects on the synthesis and turnover of neuropeptides and neurotransmitters through modulation of nuclear gene expression (10, 11). However, little attention has been paid to the influence of thyroid status on the gene expression of neuropeptides that are involved in the brain regulation of the autonomic nervous system. Convergent evidence has established that medullary TRH synthesized in the caudal raphe nuclei plays a physiological role in autonomic regulation (12). Motoneurons regulating vagal efferent activities are located in the dorsal motor nucleus of the vagus (DMN) and the nucleus ambiguus (Amb), which mainly project to the gastrointestinal tract and thoracic organs respectively (13). TRH receptors are expressed in the DMN and Amb neurons (14). Also, both nuclei are densely innervated by TRH containing nerve terminals that, at least for those in the DMN, originate solely from TRH synthesizing neurons in the raphe obscurus (Rob), raphe pallidus (Rpa) and the parapyramidal regions (15). Microinjection of TRH or the stable TRH analog into the DMN or Amb induces vagal-dependent cardiac and gastrointestinal responses, including bradycardia (16) and stimulation of gastrointestinal secretion and motility (12). Activation of raphe cell bodies by microinjection of kainic acid (16, 17) or glutamate (18, 19) into the Rpa or Rob induces similar vagal-mediated cardiovascular and gastrointestinal responses (12). Cold exposure, which increases pro-TRH messenger RNA (mRNA) in the Rpa and Rob (20), also results in vagal dependent stimulation of gastric acid secretion and motility (21). The autonomic responses to cold stress or to chemical stimulation of the caudal raphe nuclei can be prevented by blockade of endogenous TRH action using TRH antibody injected into the cisterna magna (21), or microinjected into the DMN (17) or Amb (16), or using TRH receptor antisense oligodeoxynucleotides injected into the cisterna magna (19). Tracing studies showed that TRH synthesizing neurons in the caudal raphe nuclei also contribute dense projections to the sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord (22), where TRH acts as an excitatory neurotransmitter (23). Taken together, these findings provide strong evidence that TRH-containing projections from caudal raphe nuclei to the vagal and sympathetic preganglionic motoneurons play an important role in brain stem regulation of the peripheral autonomic nervous system.

The negative feedback regulation of TRH gene expression by thyroid hormones has been well documented in neurons of the medial paraventricular nucleus (PVN) of the hypothalamus (24–26). The simultaneous occurrence of increased pro-TRH mRNA and TRH prohormone in these neurons following thyroidectomy indicate that hypothyroidism may enhance both transcription and translation of the TRH prohormone (25, 26). In contrast, TRH gene expression in other hypothalamic nuclei or thalamic sites containing TRH synthesizing neurons is not altered by hypothyroidism or hyperthyroidism (24, 25, 27). Although the medullary caudal raphe nuclei contain the most abundant group of TRH synthesizing neurons outside of the hypothalamus (20, 25), the influence of thyroid hormones on TRH gene expression in the medullary Rpa and Rob is still unknown. The aim of the present study was to determine whether alterations of circulating thyroid hormone levels induced by surgical thyroidectomy with or without T4 replacement influence TRH gene expression in the medullary caudal raphe nuclei in rats. Also, because hypothalamic TRH gene expression is suppressed by fasting (28) and medullary TRH is known to be involved in the regulation of feeding related gastric secretion and motility (29), the influence of hypothyroidism on medullary TRH gene expression was compared in both 24 h fasted and normally fed rats.

Materials and Methods

Animals

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 h). All animals were killed between 1400 and 1700 h. A previous study indicated that circadian variations in pro-TRH mRNA levels in the PVN are minimal between 1400 and 1800 h (30). Animal protocols were approved by the Veteran Administration Medical Center/West Los Angeles Research Service Animal Committee.

Thyroidectomy

Rats were anesthetized with an ip injection of a 3:1 volume mixture of ketamine (75 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (5 mg/kg; Mobay Corporation, Shawnee, KS). Before extirpating the thyroid gland, the anterior and posterior thyroid arteries were ligated, and the external parathyroid glands were carefully isolated from the thyroid gland with a fine glass needle and kept intact during extirpation of the thyroid gland. Sham operations were performed under the same conditions except that the thyroid and parathyroid glands as well as the blood supply to the glands were kept intact. Rats were awake within 15 min after the surgery and kept in individual cages for at least one week. Drinking water for thyroidectomized rats was supplemented with 2% calcium gluconate to maintain calcium homeostasis.

Experimental protocols

Rats were either sham-operated and ip injected with saline or T4 (10 or 100 μg/100 g for in situ hybridization or Northern blot analysis, respectively) (Sigma Chemical Co., St. Louis, MO) or surgically thyroidectomized and injected with saline or T4 (2 or 4 μg/100 g for in situ hybridization or Northern blot analysis, respectively). Saline or T4 was injected daily from days 2 to 30 after surgery. The T4 replacement doses were selected based on previous reports showing that administration of pharmacological doses of thyroid hormones inhibited the hypothyroidism-induced rise in hypothalamic pro-TRH mRNA (24–26, 31, 32). Body weights were monitored every 3 days. At the end of the treatments, rats were deprived of food but not water for 24 h and then killed. Five additional paired groups of sham-operated and thyroidectomized rats receiving daily saline injections and fasted for 24 h were killed respectively at 1, 2, 3, 4, and 5 weeks after surgery. Two groups of sham-operated and thyroidectomized rats receiving daily saline injections were kept normally fed without fasting before they were killed. In all experiments, animals were decapitated in a “one in each group-alternative” order. Trunk blood was collected for measuring T4 levels by RIA and brainstems were collected for measuring pro-TRH mRNA levels by Northern blot analysis and for locating the mRNA by in situ hybridization.

T4 RIA

Blood samples were immediately put on ice and centrifuged. The serum was kept at −80 C until assayed. Serum aliquots (10 μl) were used to measure total T4 levels with a commercial RIA kit (Amerlite Diagnostics Limited, Rochester, NY). The sensitivity of the assay ranged from 0 to 500 nmol/liter. All samples were measured in duplicate.

Pro-TRH hybridization probe

The hybridization probe was prepared as described in our previous studies (20). All reagents were purchased from Promega Corp. (Madison, WI). The 1322 bp EcoRI pro-TRH DNA fragment cloned in plasmid pUC12 (kindly provided by Dr. R. Goodman, Vollum Institute, Portland, OR) was subcloned into pGEM-3. The antisense cRNA probe was synthesized using 1 μg of SmaI-linearized plasmid DNA in transcription buffer containing DTT, each of ATP, GTP, and CTP, and 50 μCi [α−32P]UTP (for Northern blot analysis) or 100 μCi [α−35S]UTP (for in situ hybridization). The reaction was initiated by adding T7 RNA polymerase and incubated at 37 C for 60 min. The plasmid DNA was removed by ribonuclease-free deoxyribonuclease I. Radio-labeled RNA was purified by phenol/chloroform extraction and alcohol precipitation. For in situ hybridization, the probe was hydrolyzed by incubation with 0.2 m Na2CO3 and 0.2 m NaHCO3 at 60 C for 15 min to obtain fragments about 150 nucleotides in length.

The specificity of the pro-TRH probe has been validated in our previous studies (20). The addition of a 50-fold excess of cold probe simultaneously with the radiolabeled probe into the hybridization mix solution almost completely abolished the pro-TRH mRNA signal. Pro-TRH mRNA signal was also observed in the PVN using the same probe (20).

Northern blot analysis of pro-TRH mRNA

Rat brains were rapidly removed after decapitation, and the brainstems were dissected, put on dry ice immediately, and kept at −80 C until RNA extraction. The landmarks for dissection of the brain stem and the performance of Northern blot analyzes were as previously described (20). Briefly, denatured total RNA samples (20 μg) extracted (33) from control and experimental groups were separated on the same 1.2% formaldehyde-agarose gel and then were transferred to the same nylon membrane (Hybridization, 0.45 micron, 10 × 15 cm, Amersham Corp., Arlington, Heights, IL). RNA blots were prehybridized for 6–10 h and then incubated with fresh hybridization buffer in the presence of radiolabeled probe for 24–48 h at 55 C. The blots were exposed to x-ray films (XAR-5, Eastman Kodak Co., Rochester, NY) for 1–3 days at −80 C. The relative densities of the mRNA signals were measured quantitatively by using a Bio-Rad Laboratories, Inc. densitometer (model 620, Hercules, CA) and control levels were expressed as 100%. The consistency of RNA loading and transferring were assessed by rehybridizing the membrane with an 18S ribosomal RNA oligodeoxynucleotide probe (5′CGG CAT GTA TTA GCT CTA GAA TTA CCA CAG 3′) labeled with 32P-ATP by standard 5′ end-labeling techniques (34).

In situ hybridization of pro-TRH mRNA

Brains were removed after decapitation and immediately frozen with dry ice. Cryostat-cut brain stem sections (10 μm) were collected at the levels from interaural −2.80 to −4.30 mm according to the atlas of Paxinos and Watson (35). Sections were fixed in 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO)/PBS for 15 min and rinsed twice in PBS. Then the sections were transferred to a solution containing 0.25% acetic anhydride in 0.1 m triethanolamine (pH 8.0) for 10 min at room temperature followed by dehydrating through graded ethanol, delipidating in chloroform, rinsing in ethanol and air-drying. In situ hybridization was based on the procedures of Fremeau et al. (36). In brief, labeled cRNA probe (1 × 106 cpm/slide) was added to a hybridization cocktail (90 μl/slide), which consisted of 50% formamide, 600 mm NaCl, 80 mm Tris-HCl (pH 7.5), 4 mm EDTA, 0.1% sodium pyrophosphate, 0.2% SDS, 10% dextran sulfate, 100 mm dithiothreitol, 1 × Denhardt’s solution and 0.5 mg yeast transfer RNA per milliliter. Final hybridization was carried out for 24 h at 50 C. Then, slides were washed in 2 × SSC, treated with ribonuclease A to digest the unhybridized RNA, desalted in 1 × and 0.5 × SSC at 55 C and in 0.1 × SSC at room temperature. Slides were then washed in demineralized water followed by 95% ethanol and air-dried. Labeled sections were coated with liquid emulsion (Kodak NTB-2 diluted 1:1 with water), exposed for 7 weeks at 4 C, developed in D-19 developer and fixed in Kodak fixer. Sections were subsequently stained with Cresyl Violet and photographed under the microscope with light and dark fields. Silver grains were counted under the light microscope with oil immersion lens only on neurons cut across the nuclei as previously described (37). The numbers of neurons cut across the nuclei in each nucleus (Rpa or Rob) were 6–15 neurons per section and various in different caudal to rostral levels. For each rat, the number of silver grains per neuron in each nucleus represents the mean ± se of five random selected neurons (cut across the cell nucleus) per section and five sections in different caudal-rostral levels.

Statistics

Results are expressed as means ± se. Comparisons between two groups were analyzed by Student’s t test, and multiple groups were compared by one-way ANOVA followed by Duncan’s contrast. P values of < 0.05 were considered statistically significant.

Results

Effects of thyroidectomy and T4 injections on rat body weights and serum T4 levels

Sham-operated rats injected daily with saline gradually increased their body weight from 273 ± 5 g on the day of surgery to 351 ± 7 g on the 30th day after sham operation. The body weight of thyroidectomized rats injected daily with saline had a sharp drop during the first 3 days after surgery, then slowly increased but remained significantly lower than sham-operated rats throughout the 30 day period (303 ± 9 g on the 30th day, P < 0.01). The body weight of thyroidectomized rats with T4 replacement (2 or 4 μg/100 g·day for 30 days) had similar changes as thyroidectomized rats injected with saline during the first 2 weeks, but thereafter steadily increased to reach 345 ± 14 g and 332 ± 7 g, respectively, on the 30th day. The values were significantly higher than thyroidectomized rats without T4 replacement. The body weights of sham-operated rats injected with T4 at 10 μg/100 g·day were not significantly different from the euthyroid controls (350 ± 8 vs. 365 ± 4 g on the 30th day, n = 5/group), whereas those with T4 injection at the dose of 100 μg/100 g·day were lower than the euthyroid controls throughout the treatment period.

Serum T4 levels in thyroidectomized rats decreased by 75–87% compared with sham-operated controls throughout the 5-week period (Table 1). Thirty days after surgery, thyroidectomized rats injected daily with saline had a 2- to 3-fold decrease in T4 levels; T4 replacement (2 or 4 μg/100 g·day) reversed the hypothyroidism in thyroidectomized rats. Injection with T4 at 10 or 100 μg/100 g in the sham-operated rats resulted in 8- or >10-fold increases in serum T4 levels, respectively, compared with sham-operated rats injected daily with saline (Tables 2 and 3).

TABLE 1.

Time course of changes in serum T4 levels after thyroidectomy in rats

Treatmenta Serum T4 (nmol/liter)b
Weeks after surgery 1 2 3 4 5
Sham operation 35.4 ± 3.5 49.3 ± 3.1 55.8 ± 0.3 46.6 ± 2.6 41.5 ± 4.4
Thyroidectomy 8.8 ± 0.7c 9.0 ± 0.7c 7.1 ± 0.8c 19.22 ± 3.8c 8.6 ± 1.1c
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a

Rats were sham operated or thyroidectomized and serum sample were taken 1 to 5 weeks after the surgery.

b

Each value represents the mean ± se of 4–5 rats.

c

P < 0.001 compared with corresponding sham operated groups.

Table 2.

Serum T4 levels after different treatments in rats used for Northern blot analysis

Treatmentsa n Serum T4b (nmol/liter)
Surgery ip
Sham operation Saline 7 45.9 ± 3.3
Thyroidectomy Saline 9 15.0 ± 1.8c
Thyroidectomy T4 (4 μg/100 g) 8 381.3 ± 24.9d
Sham operation T4 (100 μg/100 g) 8 >500.0 ± 0.0c
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a

Surgeries were performed under ketamine/xylazine anesthesia and the daily ip injections of saline or T4 began the next day after the surgery for 30 days.

b

Each value represents the mean ± se of the indicated number of rats.

c

P < 0.001 compared with the sham operation + saline group.

d

P < 0.001 compared with the thyroidectomy + saline group.

TABLE 3.

Serum T4 levels after different treatments in rats used for in situ hybridization

Treatmenta n Serum T4b (nmol/liter)
Surgery ip
Sham operation Saline 5 30.8 ± 2.1
Thyroidectomy Saline 5 14.9 ± 1.1c
Thyroidectomy T4 (2 μg/100 g) 5 62.4 ± 27.1d
Sham operation T4 (10 μg/100 g) 5 248.0 ± 9.7c
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a

Surgeries were performed under ketamine/xylazine anesthesia and the daily ip injections of saline or T4 began the next day after the surgery for 30 days.

b

Each value represents the mean ± se of the indicated number of rats.

c

P < 0.001 compared with the sham operation + saline group.

d

P < 0.05 compared with thyroidectomy + saline group.

Effect of thyroid status on medullary pro-TRH mRNA levels

By in situ hybridization, pro-TRH mRNA signals in the coronal sections of the medulla were mostly located in the Rpa and Rob nuclei at the rostral-caudal levels of interaural −2.80 to −4.30 mm according the atlas of Paxinos and Watson (35) (Fig. 1).

FIG. 1.

FIG. 1.

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Schematic diagram showing the location of selected field in the medulla and dark view micrographs of medullary Rpa and Rob showing pro-TRH mRNA signals that hybridized with 35S-UTP labeled cRNA probe in rats of different thyroid status (treated for 30 days). Coronal sections at the level of interaural −3.72 mm (35) were exposed for 7 weeks. A, Sham operation + saline ip; B, thyroidectomy + saline ip; C, thyroidectomy + T4 ip (2 μg/100 g); D, sham operation + T4 ip (10 μg/100 g), ×85.

Northern blot analysis of total medullary RNA with the cRNA probe showed a single band of 1.6 kb, in agreement with the published size of pro-TRH mRNA (38). Figure 2 illustrates medullary pro-TRH mRNA and 18S ribosomal RNA signals in 24 h fasted rats with different thyroid statuses after 30 days of treatment. Quantitative analysis of the signals showed that thyroidectomized rats injected with saline had a 120% increase in medullary pro-TRH mRNA levels compared with sham-operated rats (Fig. 2). T4 replacement (4 μg/100 g·day for 30 days) completely inhibited the increase in medullary pro-TRH mRNA levels induced by thyroidectomy (Fig. 2). Sham-operated rats injected with T4 (100 μg/100 g·day for 30 days) showed no change in the pro-TRH mRNA levels compared with saline injected rats (Fig. 2). Time course studies indicated that medullary pro-TRH mRNA levels were significantly enhanced by 72.5% one week after thyroidectomy and reached a plateau at the third week after surgery (133% increase) that was maintained throughout the 5-week experimental period (Fig. 3).

FIG. 2.

FIG. 2.

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Effect of different thyroid statuses on medullary pro-TRH mRNA levels after 30 days treatment. Top, Northern blot of total RNA extracted from rat medulla in different treatment groups (24 h fasted) that hybridized with 32P-UTP labeled cRNA probe for pro-TRH mRNA or 32P-deoxy-ATP labeled oligodeoxynucleotide probe for 18S ribosomal RNA as indicated in the figure. Sham, sham operation; Tx, thyroidectomy; Tx + T4, thyroidetomy + T4 ip (4 μg/100 g · day); sham + T4, sham operation + T4 ip (100 μg/100 g·day). Bottom, each column represents mean ± se of the relative signal density in number of rats indicated inside the column. *, P < 0.05 compared with sham operation + saline ip group. #, P < 0.05 compared with thyroidectomy + saline ip group.

FIG. 3.

FIG. 3.

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Time course of changes in medullary pro-TRH mRNA levels after thyroidectomy. Top, Northern blot of total RNA extracted from rat medulla in sham-operated or thyroidectomized rat that hybridized with 32P-UTP labeled cRNA probe for pro-TRH mRNA or 32P-deoxy-ATP-labeled oligodeoxynucleotide probe for 18S ribosomal RNA as indicated in the figure. The numbers under each group of signals indicate the week after surgery. Sham, sham operation; Tx, thyroidectomy. Bottom, Signal density of the medullary pro-TRH mRNA after thyroidectomy. Each column represents mean ± se of the relative signal density in each group shown in the top of the figure. *, P < 0.05 compared with corresponding sham-operated rats.

In situ hybridization studies of medullary pro-TRH mRNA in 24 h fasted rats with different thyroid statuses (treated for 30 days) showed patterns of change similar to those observed in Northern analyzes (Fig. 1). The pro-TRH mRNA signals in the Rpa and Rob were about 2-fold more dense in thyroidectomized rats compared with sham-operated rats 30 days after surgery (Fig. 1, A and B). In addition, the number of silver grains per neuron was increased more than 3-fold in thyroidectomized rats compared with control (Table 4). T4 replacement (2 μg/100 g·day for 30 days) reversed the increase of pro-TRH mRNA signal in thyroidectomized rats (Fig. 1C, Table 4). A tendency to decrease pro-TRH mRNA signals was observed in hyperthyroid rats compared with euthyroid rats, however, the difference did not reach statistical significance (Fig. 1D, Table 4).

Table 4.

Number of silver grains per neuron in the Rpa and Rob in different thyroid statuses

Treatmenta Number of silver grains/neuronb
Surgery ip Rpa Rob
Sham operation Saline 61.2 ± 6.1 62.4 ± 7.5
Thyroidectomy Saline 220.0 ± 20.0c 210.0 ± 10.0c
Thyroidectomy T4 (2 μg/100 g) 55.3 ± 4.9d 51.6 ± 2.0d
Sham operation T4 (10 μg/100 g) 47.8 ± 13.4 47.6 ± 13.3
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a

Surgeries were performed under ketamine/xylazine anesthesia and the daily ip injection of saline or T4 began the next day after the surgery for 30 days.

b

Each value represents the mean ± se of five neurons/section, five secretion/rat and two rats/group.

c

P < 0.01 compared with the Sham operation + saline group.

d

P < 0.005 compared with Thyroidectomy + saline group.

Figure 4 shows the pro-TRH mRNA signal levels in sham-operated and thyroidectomized rats 30 days after surgery in both 24 h fasted or normally fed conditions. A 24 h fasting did not change pro-TRH mRNA levels in euthyroid rats but reduced significantly the magnitude of the medullary pro-TRH mRNA response to thyroidectomy. While the pro-TRH mRNA levels in 24 h fasted/thyroidectomized rats were 180% of the 24 h fasted/sham-operated controls (P < 0.02), the TRH mRNA levels of normally fed/thyroidectomized rats were 500% of the normally fed/sham-operated rats (P < 0.001) (Fig. 4).

FIG. 4.

FIG. 4.

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Medullary pro-TRH mRNA levels in sham-operated or thyroidectomized rats in both normally fed and 24 h fasted conditions. Top, Northern blot of total RNA extracted from medulla of 24 h fasted or normally fed rats that received either sham operation or thyroidectomy 30 days before. The blot was hybridized with 32P-UTP labeled cRNA probe for pro-TRH mRNA. Sham, sham operation; Tx, thyroidectomy; fasted, 24 h fasted rats; fed, normally fed rats. Bottom, Signal density of the medullary pro-TRH mRNA in groups of rats indicated in the top part. Each column represents mean ± se of the relative signal density in number of rats indicated inside the column. *, P < 0.05 compared corresponding sham-operated rats. #, P < 0.05 compared with 24 h fasted/thyroidectomized rats.

Discussion

It is well established that TRH-synthesizing neurons in the medial parvicellular division of the PVN in the hypothalamus are under negative feedback regulation by circulating thyroid hormones (24–26). However, studies so far indicate that negative regulation of TRH gene expression by thyroid hormone is highly selective to the hypophysiotropic TRH neurons in the PVN, which play an important role in endocrine regulation (24–26). Data obtained in the present study indicate that a negative regulation of TRH gene expression by thyroid hormones also takes place in the medullary caudal raphe nuclei, from which TRH-synthesizing neurons project to vagal and sympathetic motoneurons regulating autonomic nervous system outflow to viscera (12, 15, 22). Medullary pro-TRH mRNA levels assessed by Northern blot analyzes increased 1 week after thyroidectomy, reached a plateau at the third week, and maintained this high level throughout the 5-week experimental period. The onset and the range of the pro-TRH mRNA increase in the medulla are similar with the pro-TRH mRNA changes in the PVN after thyroidectomy (24–26, 39). In situ hybridization showed that the increased mRNA signals occurred in the Rpa and Rob, the same sites as previously reported to express pro-TRH mRNA (20, 25).

The difference in medullary pro-TRH mRNA levels between the sham-operated and thyroidectomized rats does not result from circadian variations (30) or cold exposure (20, 30) because rats in all groups were kept under same conditions of illumination and temperature, and were killed in a “one in each group-alternative” order. Data from the present study indicates that the stimulation of medullary TRH gene expression in thyroidectomized rats is related to the removal of the negative feedback signal exerted by thyroid hormones. First, elevated levels of medullary pro-TRH mRNA were maintained after thyroidectomy, which correlated with the sustained decrease in serum T4 levels throughout the experimental period. Second, T4 replacement inhibited the hypothyroidism-induced rise in medullary TRH gene expression. In support of this concept, we recently found immunoreactivities of thyroid hormone receptor isoforms and colocalization of prepro-TRH immunoreactivity with TRα1 mRNA or TRβ2 immunoreactivity in the neurons of the Rpa and Rob (40–42). Although the hormone replacement with ip injection of T4 at 4 μg/100 g·day in thyroidectomized rats brought serum T4 levels into the superphysiological range, there was no functional manifestation of hyperthyroidism, as shown by the constant body weight gain in the last two weeks of the treatment. Previous studies have documented that the suppression of the rise in hypothalamic TRH gene expression in hypothyroid rats to euthyroid levels required peripheral infusion of T3 doses inducing supraphysiological and hyperthyroid circulating range (at least 1.7 times of normal) (31, 43). High doses of T3 administration were also required to reverse changes in heart rate (9) and nuclear thyroid hormone receptor levels in the anterior pituitary (43) of hypothyroid animals. It was proposed that both T4 and T3 contribute to the feedback regulation of TRH biosynthesis in hypophysiotropic neurons of the PVN and T4 monodeiodination exists within the central nervous system (31, 43). Although many studies used T3 (9, 31, 43), T4 was also used to replace thyroid hormone in hypothyroid rats in previous studies (26, 32). The threshold dose at which exogenous thyroid hormone reverses the increase in medullary pro-TRH mRNA after thyroidectomy needs to be further assessed. However, in the present study, T4 replacement at the dose of 2 μg/100 g•day, which resulted in a 2-fold high serum T4 levels vs. the control, also reversed the increase in medullary pro-TRH mRNA in thyroidectomized rats as assessed by in situ hybridization.

Although most investigations on the feedback regulation of TRH gene expression in the PVN by thyroid hormones were studied using only hypothyroid animal models (24, 26, 31, 43), there are reports that hyperthyroidism further suppressed the pro-TRH mRNA levels in the PVN (25, 27). In the present study, however, medullary pro-TRH mRNA levels in hyperthyroid rats did not appear to have a significant change when assayed by Northern blot analysis, and only slightly decreased when assayed by in situ hybridization compared with euthyroid rats. The lack of a significant decrease may result from relatively low basal levels and/or insufficient sensitivity to measure changes in the present condition. Alternatively, it is also possible that medullary TRH gene expression is not sensitive to supraphysiological levels of circulating thyroid hormone. Future study is needed to assess whether medullary pro-TRH mRNA levels is influenced by hyperthyroidism under stimulated conditions that increase medullary pro-TRH mRNA levels, such as cold stress (20).

It is interesting to note that the increase in medullary pro-TRH mRNA levels in hypothyroid rats (30 days after thyroidectomy) was more remarkable in normally fed rats than in 24 h fasted rats. A 24 h fasting did not influence basal medullary pro-TRH mRNA levels in euthyroid rats. By contrast, the increase in pro-TRH mRNA levels induced by hypothyroidism was three times higher in normally fed rats compared with 24 h fasted rats. These results indicate that feeding and/or the fed state enhanced the stimulation of medullary TRH gene expression-induced by hypothyroidism. Alternatively, the regulation of medullary TRH gene expression is more sensitive to the stimulation of feeding and/or fed state in hypothyroid rats than in euthyroid rats. Recent studies have revealed that fasting induces suppression of TRH gene expression in the PVN (28). This may be due to a resetting of the set point for thyroid hormone dependent inhibition of pro-TRH biosynthesis by a fall in circulating leptin levels during fasting (28). Whether leptin is also involved in the regulation of medullary TRH gene expression needs further investigation. Another explanation for the difference of medullary pro-TRH mRNA levels between fasted and normally fed thyroidectomized rats may relate to the specific function of medullary caudal raphe nuclei. Previous observations have established that the activation of the medullary raphe-DMN TRH system is one of the important steps in central vagal activation mediating the cephalic and gastric phases of gastric acid secretion and motility during digestion (12). Enhanced TRH gene expression in the caudal raphe nuclei in response to feeding and digestion in thyroidectomized rats may provide further evidence that medullary TRH gene expression relates with gastrointestinal regulation, and that increases in TRH gene expression may have pathophysiological consequences related to gastrointestinal disorders accompanying hypothyroidism. Although the detailed mechanisms still need to be investigated, we recently obtained supportive evidence of such a relationship. Hypothyroidism induced a significant increase in the activity of gastric corpus histidine decarboxylase, the key enzyme in the synthesis of histamine. This increase was more remarkable (three times higher) in normally fed rats than in 24 h fasted rats (44). In addition, increased histidine decarboxylase activity could be induced by central injection of a TRH analog in fasted euthyroid rats (45).

In summary, pro-TRH mRNA levels are significantly elevated in the medulla from 1 to 5 weeks after surgical thyroidectomy in association with low circulating levels of T4 in rats. Conversely, the peripheral injection of T4 inhibited this response. These data, together with the main location of pro-TRH mRNA changes in the Rpa and Rob neurons, clearly support the view that in addition to the medial PVN, caudal medullary raphe nuclei are important sites where TRH gene expression is negatively regulated by thyroid hormones. Because TRH synthesizing neurons in the caudal raphe nuclei project directly to the medullary vagal motoneurons (15) and play an important role in central autonomic regulation (12, 29), the enhanced TRH gene expression in these caudal raphe nuclei may have important functional relevance to the understanding of autonomic-related visceral alterations induced by abnormal thyroid statuses.

Acknowledgments

We thank Paul Kirsch for assistance in the preparation of the manuscript.

This work was supported by NIH Grants DK-50255 (H.Y.), DK-30110 (Y.T.), and DK-41301, Pilot and Feasibility Award (to H.Y.).

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