Thyrotropin-releasing Hormone Neurons Research Articles

Association between pituitary adenylate cyclase-activating polypeptide and thyrotropin-releasing hormone in the rat hypothalamus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the hypothalamus including the paraventricular nucleus (PVN). In this study the anatomical relationship between PACAP- and thyrotropin-releasing hormone (TRH)-immunoreactive neuronal elements was investigated in the rat hypothalamus. Using a well-characterized mouse monoclonal antibody against PACAP and a rabbit polyclonal antiserum against TRH, we found numerous nerve fibers with PACAP-immunoreactivity (ir) closely apposed to TRH neurons in the PVN suggesting synaptic contacts. Electron microscopy confirmed the presence of synapses between PACAP-ir terminals and TRH-ir perikarya and various dendritic profiles as well as between PACAP-ir terminals and unlabeled perikarya and small- to medium-sized dendrites. Coexistence of the two peptides in perikarya of the PVN was limited to only a few neurons in the periventricular subdivision, but PACAP-ir and TRH-ir extensively coexisted in perikarya of the perifornical cell group, medial preoptic area, lateral hypothalamus and dorsomedial nucleus. The interactions between PACAP-containing neuronal processes and TRH neurons in the PVN raise the possibility that PACAP modulates the secretion of TRH destined for regulation of anterior pituitary TSH. The more general association between PACAP and TRH in other regions of the hypothalamus suggests a further role for PACAP as a cofactor in the function of TRH neurons.

Expression of the proprotein convertases PC1 and PC2 mRNAs in thyrotropin releasing hormone neurons of the rat paraventricular nucleus of hypothalamus

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60–70% of TRH neurons, and PC1 mRNA in 37–46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.

Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment.

This review focuses on the central regulation of thermoregulatory responses with special attention to the participation of thyrotropin-releasing hormone (TRH) in both autonomous and endocrine responses to a cold environment. Besides a direct projection of TRH neurons from paraventricular nuclei (PVN) to the median eminence, and the subsequent activation of the thyroid axis, there are direct projections from the PVN to the autonomic preganglionic neurons controlling autonomous responses. There projections convey information to peripheral targets involved in thermogenesis through the dorsal vagal complex and the spinal cord, for parasympathetic and sympathetic neurotransmissions respectively. Furthermore, cold exposure increases TRH mRNA levels in the PVN but also in dorsal motor and caudal raphe nuclei, thus providing strong evidence for a functional link between autonomous and neuroendocrine systems involved in thermoregulation. The review also focuses on neuroendocrine regulation of cold-induced TRH/TSH release associated with modifications in somatostatin release, with special reference to the participation of several central neurotransmitters (catecholamines, serotonin or GABA) or the influence of sex steroids.

Pups removal enhances thyrotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus

Previous studies have shown that lactation and suckling alter thyrotropin-releasing hormone (TRH) biosynthesis in hypothalamic paraventricular neurons. The amounts of paraventricular TRH mRNA and mediobasal hypothalamus (MBH) TRH were determined following removal of the pups to examine whether paraventricular TRH neuron activity is altered during the transition from lactation to estrous cycle. Paraventricular TRH mRNA and MBH TRH levels were determined by Northern blot analysis and radioimmunoassay, respectively. We had shown previously that after an 8-h withdrawal of the pups at mid-lactation the MBH TRH and paraventricular TRH mRNA levels are not modified. This condition was compared to one where pups were removed for 56 h, finding a significant decrease (46%, p < 0.005) of MBH TRH and a significant increase (156%, p < 0.02) of paraventricular TRH mRNA. The effect observed in the paraventricular TRH mRNA was correlated negatively with the serum corticosterone levels, a potential negative regulator of paraventricular TRH mRNA. The results were similar if a 1-h suckling period was introduced 8 h after withdrawal of the pups to induce a transient increase of corticosterone levels. The pattern of TRH mRNA was specific to the paraventricular nucleus because there was no enhancement in the preoptic area-anterior hypothalamus. In summary, our data suggest that TRH biosynthesis in paraventricular neurons is slowly adjusted after withdrawal of the pups, possibly to prepare TRH neurons to the new secretory demands of the estrous cycle.

Effects of acute ethanol administration and cold exposure on the hypothalamic-pituitary-thyroid axis

A single dose of ethanol increases cellular levels of the mRNA encoding thyrotropin-releasing hormone (TRH) in neurons of the paraventricular nucleus (PVN), but blocks the cold-induced increase in the levels of this mRNA. Because the thyrotropic response to cold is dependent upon TRH secretion, we hypothesized that ethanol uncouples the stimulus-induced regulation of TRH secretion from the stimulus-induced regulation of TRH expression. We employed two complementary strategies to test this hypothesis. The first was to determine whether ethanol alters pituitary sensitivity to TRH. Animals given a single intraperitoneal injection of ethanol (3 g/kg) that produced a blood alcohol concentration of nearly 300 mg/100 mL exhibited the same increase in circulating levels of TSH following an intravenous infusion of TRH. Thus, ethanol does not appear to alter pituitary sensitivity to TRH. Second, we tested whether ethanol blocks the cold induction of c-fos expression in TRH neurons of the PVN. Both cold exposure and ethanol induced the expression of c-fos in the PVN and in TRH neurons; the effects of cold and ethanol on c-fos expression were additive. Thus, ethanol clearly does not block the cold activation of TRH neurons.

Identification of thyroid hormone receptor isoforms in thyrotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus.

TRH gene expression in hypophysiotropic neurons of the hypothalamic paraventricular nucleus (PVN) is under regulation by thyroid hormone circulating in the bloodstream. To determine whether thyroid hormone could exert effects directly on TRH-producing neurons in the PVN, the presence of thyroid hormone receptors (TR) in these neurons was determined by double labeling immunocytochemical techniques, using specific antiserum to each of the functional TRs, TR alpha 1, TR beta 1, and TR beta 2, followed by antiserum to prepro-TRH-(25-50) as a marker for TRH neurons. In addition, the presence of the TR variant, TR alpha 2, was sought in these cells. Immunoreactive TR alpha 1 and TR beta 2 were found in the greatest percentage of TRH neurons in the PVN (91.1 +/- 2.5% and 83.8 +/- 2.1%) and intensely stained the nucleus. Immunoreactive TR beta 1 was also found in the majority of TRH neurons, but stained PVN cells only lightly compared to the other TRs. TR alpha 2 was found to coexist in only a minority of TRH neurons in the PVN and also lightly immunostained the nucleus compared to its more intense labeling in other regions of the brain. We conclude that hypophysiotropic TRH neurons contain functional TRs, and therefore, these neurons could be directly influenced by thyroid hormone. The relative paucity of TR alpha 2 in these cells could contribute to the selectivity of this population of TRH neurons to the effects of circulating levels of thyroid hormone.

TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats.

The role of thyrotropin-releasing hormone (TRH) in the dorsal vagal complex (DVC) in the acid response to excitation of raphe pallidus neurons was investigated in urethan-anesthetized rats with gastric fistula. Kainic acid (0.19 microgram/30 nl) microinjected into the raphe pallidus stimulated gastric acid secretion. The response was prevented by vagotomy. A specific polyclonal TRH antibody, 8964, was raised and characterized (50% inhibitory dose for TRH was 80 pg/ml at an antibody final dilution of 1:10(5)). The TRH antibody injected intracisternally blocked the acid response to intracisternal TRH, but not that of the TRH analogue RX-77368. The TRH antibody (0.33, 0.65, or 1.3 micrograms.100 nl-1.site-1) microinjected bilaterally into the DVC prevented dose dependently by 31, 60, and 76%, respectively, the increase in acid secretion induced by kainic acid injected into the raphe pallidus. The TRH antibody (1.3 microgram/site) microinjected into medullary sites outside of the DVC had no effect. These data indicate that excitation of raphe pallidus neurons induces a vagal-dependent stimulation of gastric acid secretion that is mediated by endogenous TRH in the DVC. TRH neurons in the raphe pallidus projecting to the DVC may have a physiological relevance in the vagal regulation of gastric function.

Molecular Mechanisms of Signal Integration in Hypothalamic Neurons

Synopsis. The purpose of this paper is to describe our studies focused on the mechanisms by which hypothalamic neurons process multiple signals and produce an integrated response. We illustrate our research strategy by reviewing our work on two separate neural systems: the hypo? thalamic paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN). We have focused on different peptidergic subpopulations within these nuclei to address two issues. In the PVN, we concentrate on the population of neurons containing thyrotropin-releasing hormone (TRH). These neurons are inhibited by thyroid hormones, but activated by cold exposure. Using a molecular approach, we have demonstrated that these conflicting signals simultaneously act on the same population of TRH neurons. This system will continue to be a productive model to study the mechanisms by which neurons process multiple signals. In the SCN, we concentrate on the population of neurons containing vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin releasing peptide (GRP). We have demonstrated that injection of all three peptides into the SCN of hamsters mimics the phase-delaying effects of light on circadian wheel running behavior. In addition, the genes encoding these peptides exhibit different 24-hour profiles of changes in neurons of the SCN. These data support the hypothesis that one mechanism by which these neurons produce an integrated response is by changing the concen? tration ratio of co-released peptides.

Cold- and ethanol-induced hypothermia reduces cellular levels of mRNA-encoding Thyrotropin-Releasing Hormone (TRH) in neurons of the preoptic area

Thyrotropin-releasing hormone (TRH)-containing neurons have been implicated in the central control of body temperature. TRH-containing neurons are located in brain areas known to influence body temperature, and TRH injected into these areas can produce changes in body temperature. While these lines of evidence support the view that central TRH is involved in thermoregulation, it has been difficult to confirm that TRH-containing neurons of the preoptic area are involved in this process. We used a different approach to test this hypothesis, based on recent evidence that changes in cellular levels of neuropeptide mRNA are linked to changes in neurosecretory processes. Hence, we predicted that if TRH neurons of the preoptic area are involved in body temperature regulation, cellular levels of TRH mRNA would be altered in animals in which body temperature had been experimentally altered. TRH mRNA levels were measured by in situ hybridization histochemistry in neurons of the preoptic area (POA) of animals that had been exposed to cold (5°C) or that had been given a hypothermic dose of ethanol. Cellular levels of TRH mRNA were reduced by both treatments. However, cellular levels of the mRNA-encoding gastrin-releasing peptide were not affected by these treatments in neurons of the POA, indicating that hypothermia exerted selective effects on TRH neurons in this brain region. Considering that both cold exposure and ethanol administration increase blood pressure, that the POA contains neurons which are both thermosensitive and barosensitive, and that TRH has been implicated in the control of blood pressure, we manipulated arterial blood pressure pharmacologically without changing body temperature to determine whether TRH neurons were also responsive to cardiovascular changes. Infusions with either nitroprusside, a vasodilator, or phenylephrine, a vasoconstrictor, produced significant changes in arterial blood pressure and heart rate, but did not affect TRH mRNA in the POA. These findings demonstrate that TRH neurons of the POA are thermoresponsive, supporting the view that they play a role in the central control of body temperature.

Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus

In order to investigate the possible involvement of corticotropin-releasing factor (CRF) and somatostatin (SRIF) on thyrotropin-releasing hormone (TRH) neuronal cell activity in the rat hypothalamic paraventricular nucleus, we have proceeded to the simultaneous localization of CRF or SRIF and TRH. For this purpose, we used a dual immunostaining procedure that employed antibodies to CRF and SRIF and peroxidase-labeled goat anti-rabbit IgG as a first sequence, and antibodies to a cryptic fragment (Phe 178-Glu 199) of pro-TRH (to label TRH neurons) and alkaline phosphataselabeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive CRF and SRIF fibers was observed. A large number of CRF and SRIF nerve endings were seen intimate anatomic proximity and often appeared to surround TRH-containing cell bodies. These results strongly suggest that TRH neurons might be regulated by both CRF and SRIF. These interactions might be the neuroanatomical basis for the already observed inhibitory effects of CRF and SRIF on TRH release.

Anatomical interactions of proopiomelanocortin (POMC)-related peptides, neuropeptide Y (NPY) and dopamine β-hydroxylase (DβH) fibers and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus

In order to determine the nature of afferent fibres contacting thyrotropin-releasing hormone (TRH)-synthesizing neuronal cell bodies in the rat hypothalamic paraventricular nucleus, we used dual immunostaining procedures which employed antibodies to ACTH (to label proopiomelanocortin (POMC) neurons), neuropeptide Y (NPY) and dopamine-β-hydroxylase (DβH) and peroxidase-labeled goat anti-rabbit IgG as a first sequence and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive POMC, NPY and DβH fibres was observed. Numerous NPY and POMC fibres were in intimate anatomic proximity and often appeared to surround in remarkable density TRH-containing cell bodies. Less frequent appositions between DβH fibres and TRH cell bodies were detected. These results strongly suggest that TRH neurons might be regulated by POMC, NPY as well as adrenergic and/or noradrenergic systems. These interactions might be the neuroanatomical basis for the already observed effects of opiate peptides, NPY and catecholamines on TSH secretion.

Ultrastructural analysis of the innervation of TRH-immunoreactive neuronal elements located in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus

A combination of electron microscopic immunocyotchemistry and autoradiography was employed to examine the synaptic organization of thyrotropin-releasing hormone (TRH) neurons in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus. THR neurons were identified by immunocyotchemistry. Selective uptake of tritriated serotin (5-HT) was used to identify serotininergic elements. TRH-immunoreactive axon terminals were found to be in synaptic contact with TRH-immunoreactive dendrites and with unlabeled dendritic branchets. There were direct appositions between radiolabeled 5-HT terminals and TRH-immunoreactive dendrites, but differential synaptic contacts between 5-HT axonal elements and THR neurons were not seen. TRH-immunopossitive cell bodies and dendrites received a very intense innervation by unlabeled axon terminals or axonal varicosities showing morphologically defined synaptic junctions. These were mostly of the symmetrical variety and different types could be distinguished. The findings substantiate the view that TRH neurons of the periventricular subvision of the paraventricular nucleus may be influenced by TRH axons, serotoninergic fibers and a large number of unidentified nerve terminals.

Distribution and Coexistence of Corticotropin-Releasing Factor-, Neurotensin-, Enkephalin-, Cholecystokinin-, Galanin- and Vasoactive Intestinal Polypeptide/Peptide Histidine Isoleucine-Like Peptides in the Parvocellular Part of the Paraventricular Nucleus

The distribution of several peptides, corticotropin-releasing factor (CRF), neurotensin (NT), enkephalin (ENK), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), cholecystokinin (CCK), thyrotropin-releasing hormone (TRH) and galanin (GAL) was studied in detail with immunohistochemistry in the various subdivisions of the parvocellular part of the rat hypothalamic paraventricular nucleus (PVN). Using a double-staining method and elution-restaining technique, the coexistence of CRF- and NT-like immunoreactivities (LI) with other neuropeptides was analyzed. Our results indicate that coexistence of two or more peptides in the PVN is common, and revealed that about 30% of the CRF neurons contain NT-LI and about 20% ENK-LI, whereas other peptides only occur in small fractions of the CRF cells. Thus, it seems possible to define three major subpopulations of CRF neurons, one containing NT-LI, another one containing ENK-LI and a third one apparently lacking these peptides. Conversely, about 60% of both NT- and ENK-immunoreactive neurons lacked CRF-LI. A large proportion of the small population of VIP/PHI neurons contained NT-LI. TRH neurons represented a neuron population completely distinct from the CRF neurons. Also, it did not seem to contain any of the other peptides studied with the rare exception of ENK-LI. Neuropeptides present in the PVN and presumably in nerve fibers of the external layer of the median eminence may participate in the control of the anterior pituitary hormone secretion. Whereas the role of CRF and TRH is well established, the physiological role of the other peptides studied here is still unclear.

Immunocytochemical delineation of thyrotrophic area: origin of thyrotropin-releasing hormone in the median eminence.

To identify the specific thyrotropin-releasing hormone (TRH)-containing neurons projecting to the median eminence (ME), a retrograde tracing method with horseradish peroxidase (HRP) was combined with immunocytochemical staining for TRH. Three days after HRP injection restricted to the ME, several TRH-positive neuronal perikarya were found to contain HRP. Such double-stained cells were exclusively distributed in the anterior parts of the periventricular nucleus and the most medial parts of the paraventricular nucleus. Few double-stained cells were observed in other parts of the brain examined. The present observations appear to demonstrate that the specific TRH neurons projecting to the ME are located along the border of the third ventricle, anterior to the ME.

Increase of thyrotropin-releasing hormone immunoreactivity in the nucleus of the solitary tract following bilateral lesions of the hypothalamic paraventricular nuclei

The effects of bilateral electrolytic lesions of hypothalamic paraventricular nucleus on thyrotropin-releasing hormone (TRH)-immunoreactive fibers of the nucleus of the solitary tract were studied by both immunocytochemistry and radioimmunoassay. Contrasting with a near disappearance of TRH immunoreactivity in the median eminence, both morphological and biochemical approaches demonstrate that such hypothalamic lesions induced significant increase of TRH immunoreactivity in the nucleus of solitary tract. These results confirm that TRH fibers of the nucleus of the solitary tract do not originate in the hypothalamic paraventricular nucleus (PVN). They further indicate that these TRH neurons projecting to the nucleus of the solitary tract are strongly influenced by neurons located within the PVN area.

Amyotrophic lateral sclerosis: thyrotropin-releasing hormone and histidyl proline diketopiperazine in the spinal cord and cerebrospinal fluid.

In spinal cords from seven amyotrophic lateral sclerosis (ALS) patients and four controls, we found no difference in thyrotropin-releasing hormone (TRH) concentration relative to protein content, but there was a reduction per tissue wet weight in ALS. Immunohistochemical localization of TRH in ALS cord was unaltered. Histidyl proline diketopiperazine (HisPro-DKP), a possible metabolite of TRH, was significantly elevated per protein content in ALS. CSF levels of TRH and HisPro-DKP were unchanged. These findings suggest that TRH neurons are not primarily affected in ALS, but TRH and tissue protein are lost together as the disease progresses.

Electron-microscopic cytochemistry of the catecholaminergic innervation of TRH neurons in the rat hypothalamus.

The catecholaminergic innervation of thyrotropin-releasing hormone (TRH) neurons was examined by use of a combined method of 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of 3H-noradrenaline (3H-NA) and immunocytochemistry for TRH in the same tissue sections at the electron-microscopic level. TRH-like immunoreactive nerve cell bodies were distributed abundantly in the parvocellular part of the paraventricular nucleus (PVN), in the suprachiasmatic preoptic nucleus and in the dorsomedial nucleus of the rat hypothalamus. In the PVN, a large number of immunonegative axon terminals were found to make synaptic contact with TRH-like immunoreactive cell bodies and fibers. In the combined autoradiography or 5-OHDA labeling with immunocytochemistry, axon terminals labeled with 3H-NA or 5-OHDA were found to form synaptic contacts with the TRH immunoreactive nerve cell bodies and fibers. These findings suggest that catecholamine-containing neurons, probably noradrenergic, may innervate TRH neurons to regulate TRH secretion via synapses with other unknown neurons in the rat PVN.

Thyrotropin-releasing hormone-immunoreactive neurons project from the ventral medulla to the intermediolateral cell column: Partial coexistence with serotonin

Projections from medullary thyrotropin-releasing hormone (TRH) containing neurons to the intermediolateral cell column (IML) of the thoracic spinal cord were studied in the rat. Lesions of the ventral medullary reticular formation nuclei, nucleus paragigantocellularis lateralis and nucleus interfascicularis hypoglossi, decreased the thyrotropin-releasing hormone immunoreactivity in the IML. The ventral horn and dorsal horn contents of TRH were also reduced in rats with nucleus paragigantocellularis lateris lesions. Coexistence of spinal cord TRH and serotonin was evaluated and quantified in 5, 7-dihydroxytryptamine-treated rats. Treatment with the serotonin neurotoxin reduced the TRH content of the IML by 45% and of the ventral horn by 92%. These data show that TRH containing neurons project from the ventral medulla to IML and that approximately one-half of these TRH neurons are also serotonergic. Comparisons of the effects of the same lesions on the substance P and TRH content of the IML show that neither the origin of the SP and TRH neuronal projections to the IML, nor their coexistence with serotonin, are identical.

Origin of septal thyrotropin-releasing hormone in the rat.

Through the combined demonstration of retrograde transport of horseradish peroxidase (HRP) and immunocytochemical staining of thyrotropin-releasing hormone (TRH), the TRH neurons that project to the lateral septum were identified. Following the injection of HRP into the lateral septum, retrogradely labelled neurons were detected in the bed nucleus of stria terminalis (BNST), the ventral pallidum, the anterior commissural nucleus, the lateral preoptic nucleus and the perifornical region. Some neurons in the BNST and the perifornical region were found to contain both TRH and HRP. In contrast, no TRH-containing neurons were labelled with HRP in the paraventricular nucleus, the putative thyrotropic area. These data suggest that TRH neurons projecting to the lateral septum were different from those which send fibers to the median eminence.

Early postnatal administration of 5,7-dihydroxytryptamine: Effects on substance P and thyrotropin-releasing hormone neurons and terminals in rat brain

Early postnatal administration of 5,7-dihydroxytryptamine: Effects on substance P and thyrotropin-releasing hormone neurons and terminals in rat brain