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. Author manuscript; available in PMC: 2021 May 3.
Published in final edited form as: Neurosci Lett. 1999 Dec 10;276(3):189–192. doi: 10.1016/s0304-3940(99)00830-7

Hyperthyroidism decreases thyrotropin-releasing hormone gene expression in the caudal raphe nuclei and the parapyramidal regions in rats

Pu-Qing Yuan a,b, Hong Yang a,b,*
PMCID: PMC8091821  NIHMSID: NIHMS1695517  PMID: 10612637

Abstract

Altered thyroid statuses are associated with autonomic disorders. Medullary thyrotropin-releasing hormone (TRH) and substance P (SP) regulate autonomic nervous activity. The influences of thyroid statuses on TRH and SP gene expressions in the caudal raphe nuclei and the parapyramidal regions were studied using quantitative in situ hybridization histochemistry. In male rats thyroidectomized (Tx) for 30 days, the serum T4 levels decreased by 64% and the medullary pro-TRH mRNA signals (silver grains per neuron) significantly increased by 32–45%. These changes were prevented by daily i.p. T4 (2 μg/100 g) injection in Tx rats. In sham operated/T4 (20 μg/100 g, daily) injected rats, T4 levels significantly increased by 88% and the silver grains decreased by 38–40%. Medullary SP mRNA signals were not significantly changed by altering thyroid status. These results support the concept that thyroid hormone regulates medullary TRH gene expression by negative feedback.

Keywords: Autonomic disorders, Hypothyroid, Substance P, In situ hybridization, Thyroid hormones, Feedback regulation

Altered thyroid statuses are associated with significant cardiovascular and gastrointestinal disorders [11,12]. The central mechanisms responsible for these disorders are poorly understood. Recent studies revealed that the thyroid hormone regulation of the synthesis of specific neuropeptides, which participate in the central regulation of autonomic nervous activities, may play a role [18,20]. The medullary dorsal motor nucleus of the vagus (DMN) and the nucleus ambiguus (Amb) are the main sources contributing to the vagal efferent projections to the gastrointestinal tract and the thoracic organs, respectively [6]. The medulla also contains the most abundant groups of thyrotropin-releasing hormone (TRH) synthesizing neurons outside of the hypothalamus, which are located in the raphe nuclei, including the raphe pallidus (Rpa) and the raphe obscurus (Rob), and the parapyramidal regions (PPR) [4]. Some of these neurons also synthesize substance P (SP) [4]. These neurons project to the dorsal vagal complex (DVC) including the DMN [8], which are heavily innervated with TRH and SP-containing terminals [6] and present TRH and SP receptors [2]. Microinjection of TRH into the DVC or the Amb induces vagal-dependent cardiac and gastrointestinal responses, such as bradycardia [15] and stimulation of gastric acid secretion and motility [14]. Chemical activation of the raphe cell bodies produces similar responses by releasing endogenous TRH into the DVC and Amb, which could be prevented by TRH antibody microinjected into the DVC and Amb [14,15] or TRH receptor antisense oligodeoxynucleotides injected into the cisterna magna [13]. SP modulates the vagal-mediated gastric response induced by TRH in the DVC [16]. The gene expression of medullary TRH is enhanced by certain stimuli that induce vagal-dependent gastric secretion, motility and ulceration, such as cold exposure [17]. These findings show that medullary TRH/SP containing raphe/PPR-DMN/Amb pathways play an important physiological role in the central regulation of vagal efferent activity [14]. Based on these findings, we hypothesized that thyroid hormone may regulate autonomic functions by influencing medullary TRH gene expression through a feedback mechanism similar to that established for hypothalamic TRH. We recently reported that hypothyroidism induced by thyroidectomy increases TRH gene expression in the Rpa and the Rob [18]. However, the effect of hyperthyroidism on medullary TRH gene expression could not be determined in that study due to the insensitivity of the methods used. Fos is an inducible transcription factor that may be involved in mediating TRH gene expression induced by stimuli [7]. We recently found a remarkably stimulation of Fos induction by hypothyroidism in medullary TRH synthesizing neurons, which not only appeared in the Rpa and the Rob but also in the PPR [20]. In the present study, we used quantitative in situ hybridization to further extend the evidence supporting the feedback regulation of thyroid hormone on medullary TRH gene expression. We focused on: (1) whether hyperthyroidism reduces pro-TRH gene expression; (2) whether abnormal thyroid statuses influence pro-TRH mRNA levels in the PPR, and (3) whether the thyroid hormone regulation on medullary pro-TRH gene expression is peptide selective.

Male Sprague-Dawley rats weighing 270–320 g (Harlan, CA) were either sham-operated and received daily i.p. injection with vehicle (0.02 N NaOH/saline) or T4 (20 μg/100 g, Sigma Chemical Co., ST Louis, MO) for 30 days, or surgically thyroidectomized and injected with vehicle or T4 (2 μg/100 g). Drinking water for thyroidectomized rats was supplemented with 2% calcium gluconate. Rats were deprived of food, but not water, for a 24-h period before being decapitated. Trunk blood was collected for measuring T4 levels by radioimmunoassay using a commercial kit (ICN Biomedicals, Inc, Costa Mesa, CA) and brain-stems were collected for measuring pro-TRH mRNA and SP mRNA levels by quantitative in situ hybridization.

The probe preparations and the in situ hybridization procedures were as previously described [17,18]. Briefly, the 1322 bp EcoRI pro-TRH DNA fragment (kindly provided by Dr. R. Goodman, Vollum Institute, Portland, OR) was subcloned into plasmid pGEM-3. The antisense cRNA pro-TRH probe was synthesized using 1 μg SmaIlinearized plasmid DNA as the template. The SP cRNA probe was generated using SP specific cDNA segment (560 bp, kindly provided by Dr. Paul Micevych, Department of Neurobiology, UCLA) inserted into plasmid pGEM-1 and linearized with Hind III as the template. [35S]-UTP (DuPont-New England Nuclear)-labeled probes were transcribed with T7 polymerase. Cryostat-cut brain-stem sections (10 μm) at the levels from interaural −2.80 to −4.30 mm (middle parts of the Rpa, Rob and the PPR) [10] were collected on slides and fixed in 4% paraformaldehyde (Sigma Chemical Co., St Louis, MO)/phosphate buffered saline. After the slides were treated with 0.25% acetic anhydride solution in 0.1 M triethanolamine followed by dehydrating, delipidating and air-drying, labeled probes (1 × 106 cpm/slide) diluted in a hybridization cocktail were separately applied to slides (90 μl/slide). The hybridization was carried out at 50°C for 24 h. Then the slides were washed in SSC, treated with ribonuclease A, re-washed in demineralized water followed by 95% ethanol and air-dried. Labeled sections were coated with liquid emulsion and exposed for 10 weeks at 4°C. Silver grains were counted under the light microscope with oil immersion lens [9]. The number of silver grains per neuron (Fig. 1) in each brainstem nucleus represents the mean of five random selected neurons (cut across the center of the cell nucleus) per section and five sections per rat.

Fig. 1.

Fig. 1.

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High magnification of brightfield view showing neurons with pro-TRH mRNA signals (arrows indicated) and neurons without the mRNA signals (arrow heads indicated) in the raphe pallidus. Scale bar, 25 μm.

Results are expressed as means ± SE. Comparisons among multiple groups were performed by one way analysis of variance followed by Duncan’s contrast. P-values less than 0.05 were considered statistically significant.

The serum T4 levels in different groups at 30 days after the treatment are shown in Table 1. By in situ hybridization, pro-TRH mRNA and SP mRNA signals in the medullary coronal sections at the selected levels (interaural −2.80 to −4.30 mm [10]) were mainly located in the Rpa, Rob and the PPR nuclei (Figs. 2 and 3). The pro-TRH mRNA signals in these nuclei were about one to two-fold denser and the numbers of silver grains per neuron were increased significantly by 32–45% in thyroidectomized/vehicle-treated rats (Figs. 2B and 4) compared with sham operated/vehicle rats (Figs. 2A and 4) at 30 days after the surgeries. T4 replacement (2 μg/100 g/day i.p. for 30 days) reversed the increase of pro-TRH mRNA signal in thyroidectomized rats (Figs. 2C and 4). In contrast, the pro-TRH mRNA signals in these nuclei declined significantly and the numbers of silver gains per neuron decreased by 38–40% in sham operated/T4 (20 μg/100 g per day) treated rats (Figs. 2D and 4) compared with the controls (Figs. 2A and 4). The numbers of SP mRNA silver grains per neuron in the Rpa, Rob and the PPR showed weaker changes in the same tendency with pro-TRH mRNA but were not significantly different among different thyroid statuses (Table 1).

Table 1.

Substance P mRNA levels in the Rpa, Rob and the PPR in different thyroid statuses

Treatments
 Serum T4
(μg/dl)		 Number of silver grains/neurona
Surgery i.p. (Daily for 30 days) Rpa Rob PPR
Sham operation Vehicle 4.51 ± 0.23 36.0 ± 6.5 38.6 ± 8.3 37.4 ± 7.2
Thyroidectomy Vehicle 1.61 ± 0.37* 49.7 ± 20.1 47.2 ± 15.4 47.9 ± 15.3
Thyroidectomy T4 (2 μg/100 g) 6.26 ± 0.45# 32.2 ± 7.7 33.2 ± 6.2 32.8 ± 7.3
Sham operation T4 (20 μg/100 g) 8.48 ± 1.17* 27.7 ± 9.6 29.7 ± 9.1 29.1 ± 5.3
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a

Mean ± SE of four to five rats/group.

*

P < 0.05 compared with sham operation/vehicle group.

#

P < 0.05 compared with thyroidectomy/vehicle group.

Fig. 2.

Fig. 2.

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Darkfield view micrographs (interaural −3.72 mm [10]) showing pro-TRH mRNA signals in medullary raphe pallidus (Rpa), raphe obscurus (Rob) and the parapyramidal regions (PPR) in rats with different thyroid statuses. (A) Sham operation/vehicle (i.p. daily for 30 days); (B) thyroidectomy/vehicle; (C) thyroidectomy/T4 (2 μg/100 g); (D) sham operation/T4 (20 μg/100 g). Scale bar, 80 μm.

Fig. 3.

Fig. 3.

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Darkfield view micrograph of the coronal medullary section at the level of interaural −3.72 mm [10] showing the locations of substance P mRNA signals in the Rpa, Rob and the PPR in rats. Scale bar, 90 μm.

Fig. 4.

Fig. 4.

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Influence of thyroid status on medullary pro-TRH mRNA levels. Each column represents mean ± SE of the numbers of rats indicated in the bottom of the column. Sham, sham operation; Tx, thyroidectomy. *P < 0.05 compared with sham operation/vehicle group; #P < 0.05 compared with thyroidectomy/vehicle group.

The present study confirmed our previous findings that medullary pro-TRH gene expression is increased in the hypothyroid condition [18]. In addition, we obtained evidence showing that hyperthyroidism results in a decrease of pro-TRH mRNA levels in the Rpa, Rob and the PPR, that was not achieved in the previous study [18]. Hyperthyroidism induces severe autonomic disorders characterized as a symptom of sympathetic superiority (relatively weakened vagal activity) in contrast to those induced by hypothyroidism [5,11,12]. However, direct evidence showing that high levels of thyroid hormone alter neuronal functions related to autonomic regulation is still lacking. In hypothyroidism, a significant enhancement of medullary pro-TRH gene expression, tested by Northern blot analysis, and a remarkable induction of Fos immunoreactivity in the neurons of the Rpa, Rob and the PPR were observed [18,20]. However, these parameters were not sensitive for testing the opposite changes-induced by hyperthyroidism due to the already very low basal levels in the euthyroid rats [18,20]. The present findings showed for the first time that hyperthyroidism significantly influences the function of medullary neurons participating in the regulation of vagal activity. These data also extended the evidence to support the concept that thyroid hormone exerts a feedback regulation on medullary TRH gene expression.

The influence of thyroid hormone on medullary pro-TRH gene expression is not limited to the neurons in the caudal raphe nuclei ([18]; present study), but also includes those in the PPR. The PPR consists of neurons located close to the ventral surface of the medulla and lateral to the pyramidal tract [3]. These neurons share similar neurochemical characteristics and efferent projections to the vagal motoneurons with those in the Rpa and Rob [3,8]. While the role of caudal raphe nuclei in the regulation of autonomic functions has been intensively studied [14], the functional evidence supporting a role for the PPR neurons in these regulations was obtained recently. Chemical stimulation of the PPR neurons induced vagal-dependent gastric acid secretion, gastric protection against ethanol injury and bradycardia [19]. Stimuli inducing viscera pathophysiological changes such as cold exposure and hypothyroidism, induce Fos expression in the TRH synthesizing neurons in both the caudal raphe nuclei and the PPR [1,20]. The present results showing that altered thyroid statuses influence pro-TRH gene expression in the PPR, like that observed in the Rpa and Rob, provide further evidence supporting the functional similarities of TRH-synthesizing neurons in these locations and the involvement of PPR in the medullary regulation of autonomic functions.

SP is co-localized with TRH in the Rpa, Rob and the PPR [4] and modulates TRH action on stimulating gastric secretion in the DVC [16]. However, SP mRNA levels in these medullary nuclei did not change significantly with altered thyroid statuses, indicating that the feedback regulation of thyroid hormone on medullary TRH gene expression is peptide selective.

Acknowledgments

This work was supported by NIH grants Nos. DK-50255 (H.Y.) and DK-41301 (CURE Animal Core). We thank Paul Kirsch for assistance in the preparation of the manuscript.

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