Neuropeptide Thyrotropin-releasing Hormone Research Articles

Prepro-thyrotropin releasing hormone expressing neurons in the juxtaparaventricular region of the lateral hypothalamus are activated by leptin and altered by prenatal glucocorticoid exposure

The neuropeptide thyrotropin-releasing hormone (TRH) is recognized to play an important role in controlling energy balance through direct effects on the CNS, although mechanisms explaining the phenomenon are poorly understood. To begin to understand the effects of TRH on CNS control of energy balance, we first mapped neurons expressing the TRH precursor peptide, prepro-TRH (ppTRH) in the paraventricular nucleus of the rat hypothalamus and the surrounding regions. We identified a population of ppTRH-expressing neurons in the juxtaparaventricular region of the lateral hypothalamus (LHAjp) which were stimulated by the satiety signal leptin (2.5μg/kg, IP). Using a model of fetal glucocorticoid (GC) exposure in which pregnant rats were treated with the synthetic GC dexamethasone (DEX) during gestational days 18–21, it was observed that such exposure resulted in reduced numbers of ppTRH-ir neurons in the LHAjp in adult male and female rats, and was accompanied by increased food intake. Our data provide further insight into the biological role of the LHAjp, as well as the potential involvement of TRH neurons within this region in metabolic disease associated with fetal glucocorticoid exposure.

Short-term cold exposure activates TRH neurons exclusively in the hypothalamic paraventricular nucleus and raphe pallidus

The neuropeptide thyrotropin releasing hormone (TRH) is necessary for adequate cold-induced thermogenesis. TRH increases body temperature via both neuroendocrine and autonomic mechanisms. TRH neurons of the hypothalamic paraventricular nucleus (PVN) regulate thermogenesis through the activation of the hypothalamic–pituitary–thyroid axis during cold exposure. However, little is known about the role that TRH neurons play in mediating the sympathetic response to cold exposure. Here, we examined the response of TRH neurons of rats to cold exposure in hypothalamic regions including the PVN, the dorsomedial nucleus and the lateral hypothalamus along with areas of the ventral medulla including raphe obscurus, raphe pallidus (RPa) and parapyramidal regions. Our results using a double immunohistochemistry protocol to identify TRH and c-Fos (as a marker of cellular activity) followed by analysis of preproTRH gene expression demonstrate that only TRH neurons located in the PVN and the RPa are activated in animals exposed to short-term cold conditions.

RNAi‐mediated reversible opening of the blood‐brain barrier

The blood-brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier. Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin-5 to the endothelial cells of the BBB in mice. We have shown a significant decrease in claudin-5 mRNA levels 24 and 48 hours post-delivery of siRNA, with levels of protein expression decreasing up to 48 hours post-injection compared to uninjected, phosphate-buffered saline (PBS)-injected and non-targeting siRNA-injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size-selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin-releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post-injection of siRNA targeting claudin-5 significantly modifies behavioural output. These data demonstrate that it is now possible to transiently and size-selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases.

Effect of Thyrotropin-Releasing Hormone and Its Synthetic Analogue Digipramine on Certain Indices of Hemostasis in vitro and in vivo

The effect of neuropeptide thyrotropin-releasing hormone (TRH) and its synthetic analogue digipramine on certain indices of blood coagulation and fibrinolysis were studied in vitro and in vivo. The peptides added to the pool of normal rat plasma at 10−10 to 10−3 M increased the procoagulant activity but had virtually no effect on fibrinolysis. Intravenous administration of TRH and digipramine increased the procoagulant activity of the blood and platelet aggregation but decreased fibrinolysis; digipramine had a more pronounced effect on the coagulation potential of the blood and a less pronounced effect on fibrinolytic indices as compared to TRH. Intranasal administration of the peptides did not change the pattern of their effect on indices of hemostasis although the effects became less pronounced.

Excitatory Effects of Thyrotropin-Releasing Hormone in the Thalamus

The activity of the thalamus is state dependent. During slow-wave sleep, rhythmic burst firing is prominent, whereas during waking or rapid eye movement sleep, tonic, single-spike activity dominates. These state-dependent changes result from the actions of modulatory neurotransmitters. In the present study, we investigated the functional and cellular effects of the neuropeptide thyrotropin-releasing hormone (TRH) on the spontaneously active ferret geniculate slice. This peptide and its receptors are prominently expressed in the thalamic network, yet the role of thalamic TRH remains obscure. Bath application of TRH resulted in a transient cessation of both spindle waves and the epileptiform slow oscillation induced by application of bicuculline. With intracellular recordings, TRH application to the GABAergic neurons of the perigeniculate (PGN) or thalamocortical cells in the lateral geniculate nucleus resulted in depolarization and increased membrane resistance. In perigeniculate neurons, this effect reversed near the reversal potential for K+, suggesting that it is mediated by a decrease in K+ conductance. In thalamocortical cells, the TRH-induced depolarization was of sufficient amplitude to block the generation of rebound Ca2+ spikes, whereas the even larger direct depolarization of PGN neurons transformed these cells from the burst to tonic, single-spike mode of action potential generation. Furthermore, application of TRH prominently enhanced the afterdepolarization that follows rebound Ca2+ spikes, suggesting that this transmitter may also enhance Ca2+-activated nonspecific currents. These data suggest a novel role for TRH in the brain as an intrinsic regulator of thalamocortical network activity and provide a potential mechanism for the wake-promoting and anti-epileptic effects of this peptide.

The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.

Cerebrovascular Effects of the TRH Analogues pGlu-3-methyl-His-Pro Amide and pGlu-Glu-Pro Amide: A Comparison with TRH

The goal of the study was to assess whether TRH analogues possess cerebrovascular effects similar to the native peptide. The neuropeptide thyrotropin releasing hormone (TRH) elicits cerebrovasodilation in several species under various conditions. The laser-Doppler method was employed to study the effects of TRH and the analogues pGlu-3-methyl-His-Pro amid (M-TRH) and pGlu-Glu-Pro amide. Intravenous (iv.) injection of 300 μg kg−1 of TRH elicited cerebrovasodilation and a 62% increase in blood flow within 1 minute. M-TRH, in a dose of 300 μg kg−1 iv., elicited a 80 % increase in cerebral blood flow. Even a minute dose of M-TRH (625 ng kg−1) caused an increase in cerebral blood flow. No clear difference in effects on the cerebral blood flow was observed between spontaneously and mechanically ventilated animals. pGlu-Glu-Pro amide had no cerebrovascular effect.

Hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons: histochemical relationship to thyrotropin-releasing hormone, melanin-concentrating hormone, orexin/hypocretin and neuropeptide Y

Recent demonstrations of the feeding-inhibitory properties of putative peptides derived from cocaine- and amphetamine-regulated transcript (CART) raise the question of interactions between CART peptides and other messenger molecules implicated in the control of food intake. The present study investigated the histochemical relationship of CART to the neuropeptides thyrotropin-releasing hormone (TRH), melanin-concentrating hormone (MCH), orexin/hypocretin and neuropeptide Y (NPY) in the hypothalamus. Double-label in situ hybridization showed that CART to a great extent is co-expressed with TRH in hypothalamic paraventricular nucleus neurons. This technique was also used to demonstrate that MCH, but not orexin/hypocretin, mRNA colocalized with CART in neurons of the dorsomedial hypothalamic nucleus/lateral hypothalamic area. CART-peptide immunoreactive cell bodies in this region, as well as in the arcuate nucleus and the medial posterodorsal nucleus of the amygdala, were all seen to have close appositions formed by NPY-immunoreactive nerve terminals. Lastly, in a study of mice treated with the neurotoxin, monosodium glutamate, which targets the arcuate nucleus, a near-total ablation of CART peptide immunoreactive cell bodies in this nucleus was accompanied by decreased terminal staining for CART peptide in the paraventricular hypothalamic nucleus, the arcuate nucleus itself and in the dorsomedial hypothalamic nucleus. These findings further define the position of hypothalamic CART neurons within the hierarchy of brain circuitries regulating energy balance, demonstrating the presence of CART peptide in several cell populations that form putative down-stream targets of NPY terminals, including hypophysiotropic TRH neurons and lateral hypothalamic MCH neurons.

Estrogen suppresses gastric motility response to thyrotropin-releasing hormone and stress in awake rats

Symptoms associated with gastric motility alteration vary with stress and ovarian hormone status, most notably in women with irritable bowel syndrome. This study examines combined effects, comparing gastric motility during administration of a stress-related neuropeptide thyrotropin-releasing hormone (TRH) and restraint stress in conscious rats of varied ovarian hormone status. Adult rats were ovariectomized and implanted with estrogen, progesterone, or vehicle-releasing pellets. After 21 days, intracerebroventricular (i.c.) cannula and gastric tension transducer were implanted. After 25-27 days, motility was recorded during neuropeptide injection (TRH/saline i.c.) or restraint stress. TRH induced increased motility in all groups; the response varied with hormone group, and was least and briefest in estrogen-treated rats. Motility during restraint varied with hormone group; it was diminished in estrogen-treated but not other groups. Ovarian hormone status (estrogen) modifies gut response to TRH and restraint stress.

Effect of intragastric administration of beta-endorphin on thyrotropin-releasing hormone and somatostatin release into gastric lumen of rats.

The neuropeptides beta-endorphin (BE), thyrotropin-releasing hormone (TRH), and somatostatin (SOM) in the gastric mucosa have the capacity to regulate gastric function. To clarify the possible role of BE in the interactions of neuropeptides and gastric acid secretion we investigated the effect of the intragastric administration of BE on TRH and SOM release into the gastric lumen. BE (100ng/kg) induced an immediate decrease in TRH release and a reciprocal increase in SOM release into the gastric lumen, followed by the suppression of gastric acid secretion. When various doses of BE (0-500ng/kg) were administered, changes in TRH and SOM occurred in a dose-dependent manner. Pretreatment with naloxone dihydrochloride (5 mg/kg, intraperitoneally) completely inhibited the BE-induced changes in the release of these peptides. These findings suggest that BE has a paracrine effect on TRH and SOM release into the gastric lumen through opioid receptors, and that these interactions may be involved in the regulation of gastric acid secretion.

The neuropeptide TRH has a minor effect on the enzymatic activity of acetylcholinesterase in vitro

The neuropeptide thyrotropin-releasing hormone (TRH) elicits a variety of physiological effects of which some are due to cholinergic mechanisms. TRH modulates in vivo the effects of compounds affecting acetylcholinesterase (AChE). In the present study the in vitro effects of TRH on the activity of AChE were explored. TRH has no effect at physiologically relevant concentrations. At unphysiologically high concentrations (>5 mM) a slight inhibition was found. This was noticed also when the enzyme was exposed to the amide-free tripeptide analog p-Glu-His-Pro. We conclude that any cholinergic effect of TRH observed in vivo is unlikely to be due to a direct interaction of the peptide with AChE.

Thyrotropin-releasing hormone (TRH) is markedly increased in the rat brain following soman-induced convulsions

Soman is an organophosphorus (OP) compound which irreversibly inhibits acetylcholinesterase (AChE), the primary synaptic inactivator of acetylcholine. Resultant excessive cholinergic activity elicits generalized convulsions and brain lesions. Recent evidence suggests that other neurotransmitter/neuromodulator systems may be affected by the OP compounds as well. Since we have shown that both electrically and chemically induced seizures cause significant and prolonged increases in the neuropeptide thyrotropin-releasing hormone (TRH) in epileptogenic sites, we examined soman-induced convulsion effects on CNS TRH. Rats were injected with either soman (100 μg/kg SC; equivalent to 0.9 LD 50) or saline and observed for convulsive activity. Forty-eight hours post injection, dramatic increases of TRH over control levels were seen in frontal cortex (30-fold), pooled cortex (24-fold), hippocampus (16-fold), piriform cortex (14-fold), entorhinal cortex (11-fold), and amygdala (2-fold). No change was observed in either hypothalamus or pituitary. Our results demonstrate, for the first time, a substantial effect of an OP on a specific neuropeptide system in vivo. The neurochemical and behavioral consequences of the soman-induced increases in TRH, especially in the frontal cortex, are presently unknown. Clearly, much more work is required to discern the exact role TRH has following soman exposure.

Effects of the Neuropeptide Thyrotropin-Releasing Hormone on GABAergic Synaptic Transmission of CA1 Neurons of the Rat Hippocampal Slice During Hypoxia

Barbieri, M. and A. Nistri. Effects of the neuropeptide thyrotropin-releasing hormone on GABAergic synaptic transmission of CA1 neurons of the rat hippocampal slice during hypoxia. Peptides 18(4) 585–591, 1997—Because thyrotropin-releasing hormone (TRH) has been suggested to improve recovery of brain neurons from hypoxia, which strongly impairs GABAergic synaptic transmission, the present electrophysiological study used intracellular recording from CA1 neurons of the rat hippocampal slice to examine the cellular mechanisms underlying this phenomenon. Hypoxia induced by superfusion with a medium devoid of oxygen evoked typical membrane hyperpolarization, fall in input resistance, and strong depression of monosynaptic, GABA A receptor-mediated fast inhibitory postsynaptic potentials (IPSPs). The depression of fast IPSPs during hypoxia was found to be due to a combination of factors such as shift in the IPSP reversal potential and membrane hyperpolarization. GABA B receptor-mediated slow IPSPs were comparatively less sensitive to hypoxia. TRH (10 μM), applied 1 min prior to hypoxia, selectively accelerated recovery of membrane potential and delayed return of fast IPSPs to control amplitude without changing the mechanisms responsible for depression of GABAergic transmission. In conclusion, despite a slower recovery of IPSPs, TRH facilitated earlier return of neuronal excitability after the hypoxic period.

Actions of norepinephrine on rat hypoglossal motoneurons.

1. We used conventional intracellular recording techniques in 400-microns-thick slices from the brain stems of juvenile rats to investigate the action of norepinephrine (NE) on subthreshold and firing properties of hypoglossal motoneurons (HMs). 2. In recordings in current-clamp mode, 50 or 100 microM NE elicited a reversible depolarization accompanied by an increase in input resistance (RN) in all HMs tested (n = 74). In recordings in single-electrode voltage-clamp mode, NE induced a reversible inward current (INE) accompanied by a reduction in input conductance. The average reversal potential for INE was -104 mV. The NE responses could be elicited in a Ca(2+)-free solution containing tetrodotoxin, indicating that they were postsynaptic. 3. The NE response could be blocked by the alpha-adrenoceptor antagonist prazosin, but not by the beta-adrenoceptor antagonist propranolol, and could be mimicked by the alpha 1-adrenoceptor agonist phenylephrine but not by the alpha 2-adrenoceptor agonist UK 14,304 or by the beta-adrenoceptor agonist isoproterenol when alpha-adrenoceptors were blocked. 4. Substitution of barium for calcium in the perfusion solution blocked the increase in RN in response to NE without completely blocking the depolarization. Replacement of sodium chloride with choline chloride in the barium-substituted perfusion solution blocked the remaining depolarization. 5. The neuropeptide thyrotropin-releasing hormone (TRH), which also depolarizes and increases the RN of HMs, occluded the response of HMs to NE. 6. NE altered HM firing properties in three ways: it always lowered the minimum amount of injected current needed to elicit repetitive firing, it increased the slope of the firing frequency versus injected current relation in 8 of 14 cells tested, and it increased the delay from the onset of the depolarizing current pulse to the first evoked spike in all cells tested. 7. We conclude that NE acts directly on alpha 1-adrenoceptors to increase the excitability of HMs. It does this by reducing a barium-sensitive resting potassium current and activating a barium-insensitive inward current carried primarily by sodium ions. A portion of the intracellular pathway for these actions is shared by TRH. In addition, there is evidence that NE alters HM firing patterns by affecting currents that are activated following depolarization.

Thyroid hormones rapidly and stringently regulate the messenger RNA levels of the thyrotropin-releasing hormone (TRH) receptor and the TRH-degrading ectoenzyme.

The responsiveness of adenohypophyseal target cells for the hypothalamic neuropeptide TRH is known to change depending on the hormonal and physiological conditions of the organism. We describe here the effects of thyroid hormones on the transcript levels of the TRH receptor, the TRH-degrading ectoenzyme, and TSH in rat pituitary, as revealed by Northern blot analysis. After a single injection of T3 (30 micrograms/100 g BW), the transcript levels of the TRH receptor decreased transiently, reaching 35% of control values 4 h after injection, and returned to basal levels within 24 h. In contrast, the messenger RNA (mRNA) levels of the TRH-degrading ectoenzyme increased more dramatically in response to the same hormonal stimulus. Maximal levels (> 10 times above the control value) were present from 6-24 h after the injection, returning to basal values within 96 h. For both transcripts, the observed effects changed in a dose-dependent manner, but the mRNA levels of the TRH-degrading ectoenzyme were more tightly regulated. Under the experimental conditions used, the mRNA levels of PRL and GH were not affected by the application of T3, and those of alpha-subunit exhibited only minor reductions. The TSH beta transcripts however, decreased rapidly in length and slowly in concentration, finally reaching almost undetectable levels 48 h after the injection. Subsequently, newly synthesized TSH beta mRNA, the same size as the transcripts from euthyroid rats, could be detected 96 h after treatment with T3. Upon treatment of the animals with the mild goitrogenic agent n-propylthiouracil (200 mg/liter drinking water), a fast reduction in the transcript levels of the TRH-degrading ectoenzyme became evident. Within 1 day, mRNA levels decreased to less than 50% of control values. At this stage, no effects were observed on the transcript levels of either the TRH receptor or TSH beta. After 4 days of n-propylthiouracil treatment, the mRNA levels of the enzyme decreased further to 15% of control values, whereas the transcript concentrations of the TRH receptor and TSH beta rose by factors of 2 and 3.3, respectively. The extremely stringent regulation of the TRH-degrading ectoenzyme, a mirror image of that of the TRH receptor, strongly suggests that this enzyme represents an important regulatory element, controlling the stimulation of TRH target cells and, thus, adenohypophyseal hormone secretion.

Three phases of TRH-induced facilitation of exocytosis by single lactotrophs

Membrane capacitance measurements were used to study neuropeptide modulation of exocytosis by perforated patch clamped rat lactotrophs. We report that depolarizing voltage-clamp pulses evoke exocytosis that is steeply dependent on Ca2+ influx through voltage-gated Ca2+ channels. Furthermore, we find that the neuropeptide TRH (thyrotropin-releasing hormone) acts in three phases to promote exocytosis. First, TRH transiently (within approximately 0.5 min) triggers depolarization- and extracellular Ca(2+)-independent exocytosis. Second, within 3 min of application, TRH facilitates depolarization-evoked exocytosis while inhibiting voltage-gated Ca2+ current. Finally, after 8 min, TRH further enhances depolarization-evoked exocytosis by increasing high-voltage-activated (HVA) Ca2+ channel current. Activation of protein kinase C (PKC) with a phorbol ester also stimulates depolarization-evoked exocytosis by increasing Ca2+ current. Therefore, PKC can only account for the last effect of TRH. Thus, a single neuromodulator may employ several temporally distinct mechanisms to stimulate peptide secretion.

Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone

Many neurotransmitters regulate action potential activity in neuronal, endocrine, and cardiac cells by rapidly modulating the gating of K+ channels. Neurotransmitters might also produce prolonged effects on excitability by regulating the expression of K+ channel genes. Here we show that the neuropeptide thyrotropin-releasing hormone (TRH) down-regulates Kv1.5 and Kv2.1 K+ channel mRNAs in clonal pituitary cells. The effect on Kv1.5 mRNA expression does not require protein synthesis and is due to decreased transcription. Immunoblots demonstrate that Kv1.5 and Kv2.1 immunoreactivities are significantly reduced by TRH within 12 hr. The change in channel protein expression is associated with a decrease in voltage-gated K+ currents. Thus, TRH enhances excitability by inhibiting K+ channel gene expression. Neuropeptide regulation of K+ channel gene expression may produce long-term changes in neuronal action potential activity and synaptic transmission.

Thymus-Neuroendocrine Interactions in Extrathymic T Cell Development

Studies of the development of murine intestinal intraepithelial lymphocytes (IELs) have yielded markedly different results depending on the experimental system used. In athymic radiation chimeras, IELs consist of all subsets found in euthymic mice; adult mice that were athymic at birth have only IELs that are positive for T cell receptor gamma delta and CD8 alpha alpha. These differences are resolved by the finding that administration of the neuropeptide thyrotropin-releasing hormone to adult mice thymectomized as neonates leads to the development of all IEL T cells. Thus, a neuroendocrine signal initiated by the thymus during fetal or neonatal life appears to be required for subsequent extrathymic maturation of gut alpha beta T cells.

Modulation by TRH of NMDA-elicited responses of CA1 neurones of the rat hippocampal slice preparation

Intracellular recording from CA1 neurones of the rat hippocampal slice preparation was carried out to assess the ability of the endogenously-occurring neuropeptide thyrotropin-releasing hormone (TRH) to modulate responses elicited by the excitatory amino acid agonist N- methyl- d-aspartate (NMDA). TRH (5 μM) produced no change in resting membrane potential or input resistance but facilitated on-going synaptic activity. In the continuous presence of the peptide responses to NMDA were selectively enhanced for about 20 min. In approximately 50% of cells the potentiating effect of TRH persisted in tetrodotoxin (TTX) solution and was associated with removal of the apparent voltage-dependent increase in input resistance usually found during the NMDA-induced depolarization. It is suggested that TRH evoked a transient upregulation of NMDA responses which might account for the reported facilitation by this peptide of long term potentiation in the hippocampus.

Effects of Thyrotropin- and Corticotropin-Releasing Hormone on Blood Pressure and Plasma Catecholamines in the Anesthetized Rat

The neuropeptides thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) have been found to be potent stimulators of the autonomic nervous system in both experimental animals and humans. We studied the effects of different doses of CRH and TRH given intracerebroventricularly in the urethane-anesthetized rat and a single dose of CRH in the chloral hydrate-anesthetized rat to elucidate the effects of these peptides in the unconscious state. All TRH doses studied enhanced blood pressure and noradrenaline and adrenaline secretion. Surprisingly, there were no blood pressure increases following administration of 0.4, 1.3 and 1.7 nmol CRH. In general, there was a tendency for blood pressure to decrease with the largest drop observed after 1.7 nmol CRH. Our data suggest that only TRH clearly augments blood pressure and catecholamine secretion in the anesthetized animal, while CRH does not exert major effects under the two anesthetic conditions employed.