Immunohistochemical localization of TRH in rat CNS: comparison with RIA studies

The localization of thyrotropin releasing hormone (TRH) in rat brain determined by use of avidin-biotin immunoperoxidase histochemistry was compared with the distribution and quantitation by radioimmunoassay (RIA). Male Sprague-Dawley rats received intracisternal injections of 100 micrograms of colchicine or saline and were sacrificed 24 hours later. Brains were either perfused with lysine-periodate fixative and processed for TRH immunohistochemistry or were dissected into 9 brain regions for TRH RIA. In colchicine pretreated rats. TRH immunoreactive perikarya were observed only in nuclei of the hypothalamus and brain stem. No cell body staining was observable in non-colchicine treated rats. With the exception of the olfactory bulb, brain regions exhibiting dense TRH staining contained high concentrations of TRH as measured by RIA. Colchicine pretreatment did not alter the concentration of TRH in most brain regions, however, there was a significant increase in brain stem TRH content 24 hours following colchicine administration. These findings indicate that immunohistochemical localization of TRH corresponds well with endogenous concentrations of TRH determined by RIA.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

Brain thyrotropin-releasing hormone matures independently of the hypothalamus in the rat

To evaluate the relationship of the extrahypothalamic brain thyrotropin-releasing hormone (TRH) to its hypothalamic counterpart, we studied the maturation of hypothalamic and extrahypothalamic TRH in the rat. The absolute increase of TRH in the whole brain and the extrahypothalamus reached adult levels at 7 days of age, whereas the hypothalamic TRH concentrations did not differ from the adult levels at 23 days. Moreover, the TRH concentrations at 7 days were greater than the adult levels in the striatum, hippocampus, pons-medulla and cerebellum, and similar to the adult levels in the midbrain and cortex. These data indicate the developmental divergency of hypothalamic and extrahypothalamic TRH, implying that the maturation of extrahypothalamic TRH is independent of the hypothalamus. The present study suggests that extrahypothalamic TRH may play a neurophysiological role in the central nervous system at an early infantile age, at which hypothalamic TRH is not ripe for its endocrinological action.     

Developmental changes in the distribution of rat brain pyroglutamate aminopeptidase,a possible determinant of endogenous cyclo(His-Pro) concentrations

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and Pyroglutamate aminopeptidase activity in adult and developing rat brains were studied. A comparison of the subcellular distribution of Pyroglutamate aminopeptidase activity in hypothalamic and cerebral cortical extracts from adult rats exhibited remarkable differences. In hypothalamus, the enzyme activity was mainly associated with the soluble fraction whereas in cortex it was predominantly associated with the particulate fractions. During postnatal development, the brain concentrations of cyclo(His-Pro) and Pyroglutamate aminopeptidase activities declined with age. These data suggest that Pyroglutamate aminopeptidase activity, but not TRH, plays an active role in determining the levels of endogenous cyclo(His-Pro) concentrations in brain.     

Protein-energy malnutrition during pregnancy alters caffeine's effect on brain tissue of neonate rats

We studied whether protein-energy malnutrition changed brain susceptibility to a small dose of caffeine in newborn rats. Since we had demonstrated previously that caffeine intake during lactation increased the brain neuropeptide on newborns, we investigated further the effects of the prenatal administration of caffeine on TRH and cyclo (His-Pro). From day 13 of gestation to delivery day, pregnant rats in one group were fed either a 20% or a 6% protein diet ad libitum, and those in the other group were pair-fed with each protein diet supplemented with caffeine at an effective dose of 2 mg/100 g body weight. Upon delivery, brain weight, brain protein, RNA, DNA and the neuropeptides thyrotropin-releasing hormone (TRH) and cyclo (His-Pro) were measured in the newborn rats. A 6% protein without caffeine diet caused reductions in brain weights and brain protein, RNA and DNA contents, but did not alter brain TRH and cyclo (His-Pro) concentrations in the newborn animals. In the offspring from dams fed a 6% protein diet, caffeine administration significantly elevated brain weights and brain contents of protein, RNA and DNA. In contrast, these values were similar between noncaffeine and caffeine-supplemented animals in a 20% protein diet group. Brain TRH and cyclo (His-Pro) concentrations were not changed by caffeine administration. These data suggest that caffeine augments protein synthesis in the newborn rat brain when malnourished, but that the same dose of caffeine did not affect protein synthesis in brains of newborn rats from normally nourished dams. Therefore, the present findings indicate that the nutritional status of mothers during pregnancy has important implication in the impact of caffeine on their offspring's brains.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs.

Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.     

Regional Dissociation of Cyclic AMP and Inositol Phosphate Formation in Response to Thyrotropin-Releasing Hormone in the Rat Brain

The present study was undertaken to define effects of thyrotropin-releasing hormone (TRH) on formation of cyclic AMP (cAMP) and inositol phosphates (IPs) in rat brain regions. The brain of male Wistar rats was dissected into seven discrete regions, and each region was sliced. The slices were incubated in Krebs-Henseleit glucose buffer containing varying doses of TRH. TRH caused a significant and consistent increase in cAMP level, but not in formation of IPs, in the hypothalamus, striatum, and midbrain. TRH stimulated formation of IPs in the cerebellum, where the tripeptide did not change the cAMP level. In contrast, formation of neither cAMP nor IPs was affected by TRH in the cortex, hippocampus, or pons-medulla. These data suggest that TRH possesses two distinct types of brain intracellular signaling systems, which vary with brain regions.     

Barbiturate competition for TRH receptors in mouse brain: neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.     

Molecular evolution of prepro-thyrotropin-releasing hormone in the chicken (Gallus gallus) and its expression in the brain

A cDNA encoding prepro-thyrotropin-relaesing hormone (ppTRH) in chicken (Gallus gallus) was isolated and the sites of expression in the brain were determined. The chicken ppTRH cDNA encodes 260 amino acids, including four TRH progenitor sequences (-Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg-). It is interesting to note that chicken ppTRH harbors four TRH progenitor-like sequences. According to the hydropathy profile of chicken ppTRH, not only the TRH progenitor sequences but also the TRH progenitor-like sequences are localized in hydrophilic regions. The TRH progenitor-like sequences might be related to structural conservation in the evolution of ppTRH, although they cannot be processed into TRH due to the mutation of several amino acids. According to the alignment of the deduced amino-acid sequences of known vertebrate ppTRHs and the molecular phylogenetic tree we constructed, we speculate on the molecular evolution of ppTRH in vertebrates. In situ hybridization demonstrated experession of the ppTRH gene in the nucleus preopticus periventricularis, nucleus preopticus medialis, regio lateralis hypothalami, paraventricular nucleus, nucleus periventricularis hypothalami, and nucleus ventromedialis hypothalami in the chicken brain.     

Influences of maternal caffeine on the neonatal rat brains vary with the nutritional states

The potential effect of maternal caffeine ingestion upon total brain protein and the concentration of two prototype neuropeptides, thyrotropin-releasing hormone (TRH) and its derivative, cyclo (His-Pro) in neonates was examined during the nursing period in the context of variable maternal protein intake. Maternal caffeine intake (2 mg/100 g body weight) significantly increased the total brain protein of neonates derived from dams fed a 6% casein diet, but not from dams fed a 12%- or 20%-casein diet. Maternal caffeine consumption significantly increased the amount of cyclo (His-Pro) in the neonatal brains in all groups. The percent increments in pups from dams fed 6%, 12%, and 20% casein diets were respectively 137%, 131%, and 120%. By contrast, no significant alterations were observed in TRH concentrations between caffeine and control groups. It is concluded that maternal caffeine can influence neonatal brain protein and cyclo (His-Pro) during nursing under conditions of protein-energy malnutrition.     

Selective cardiorespiratory activity of an iodinated analog of thyrotropin-releasing hormone (TRH)

The biological activity of thyrotropin-releasing hormone (TRH) and its analogs 4(5)-I-Im-TRH and 2,4(5)-I2-Im-TRH was assessed by means of their effects on: 1) the mean arterial pressure (MAP), 2) heart rate (HR), 3) ventilation minute volume (MV), 4) contractility of the rat duodenum, and 5) concentrations of thyrotropin (TSH) or prolactin (PRL) in serum. Also their binding to TRH-receptors in brain homogenates was studied. In urethane-anesthetized rats TRH ICV increased MAP, HR and MV. 4(5)-I-Im-TRH was equally as active as TRH on HR and MV but a significant elevation in MAP was observed only at a dose 100-fold to that of TRH. However, the maximal responses of 4(5)-I-Im-TRH and TRH did not differ. In conscious rats, TRH 1A elevated MAP and HR but 4(5)-I-Im-TRH was active on MAP only. 2,4(5)-I2-Im-TRH was devoid of cardiorespiratory activity. TRH dose-dependently inhibited the contractions of the rat duodenum while the iodinated analogs lacked such an activity. To induce a significant release of TSH several hundred times more of 4(5)-I-Im-TRH and over 1000 times more of 2,4(5)-I2-Im-TRH were needed as compared to TRH. The iodoanalogs elevated PRL levels only at doses 2000-fold higher than those of TRH. The iodoanalogs displaced [3H][3-Me-His2]TRH [( 3H]MeTRH) from its binding sites at concentrations about 1000 times higher than those of TRH. Substitutions of the histidyl moiety of TRH in 4(5)-I-Im-TRH and 2,4(5)-I2-Im-TRH resulted in substantial loss of the endocrine activity. While the di-iodinated analog was practically devoid of any biological activity the monoiodinated analog exerted similar cardiorespiratory activity to that of TRH.     

Studies on thyrotropin releasing hormone in cerebellar ataxia mutant mouse brain

The effects of cathecholamine on the regional TRH distribution in the brain was studied in rolling mouse Nagoya (RMN) and non-affected C3H mice. TRH was extracted from the hypothalamus, brain stem, cerebellum, and cerebrum one hour after i.p. injection of the precursor or inhibitors of cathecholamine. TRH was distributed throughout the brain of both affected and non-affected mice; however, in RMN, TRH levels were lower in the hypothalamus and higher in other areas. 1-Dopa caused a decrease of TRH in the brain stem but no change in other regions in the RMN brain, whereas it caused an increase in TRH levels in all areas of the C3H brain. Fusaric acid increased TRH in the hypothalamus of RMN and decreased it in the cerebellum; alpha-MPT also caused a decrease in the TRH level in the cerebellum. Reserpine increased the TRH level in the hypothalamus and decreased it in the cerebrum. From these results, it appears that cerebellar ataxia in RMN does not result from a decrease in the TRH, which is actually increased in the cerebellum. Catecholamine had different effects on TRH levels in RMN and the controls; this might be due to the excess accumulation of noradrenaline in the RMN brain.     

Brain TRH and Cyclo (His-Pro) and brain protein in the newborn rat are altered by maternal liquid protein feeding

Liquid protein diet (LPD) has been shown previously to produce maternal and fetal weight loss and fetal congenital anomalies, including cataracts and craniofacial malformations. Therefore, to examine the effects of LPD in pregnancy upon the central nervous system of pups, pregnant dams were fed either a 20% casein diet ad libitum, a 20% LPD, or pair-fed with a 20% casein diet. LPD was associated with significant maternal weight loss, and pups had significantly lower birth weights (5.14 +/- 0.64) than pups from the pair-fed controls (5.70 +/- 0.46, p less than 0.05). Total brain protein content was reduced significantly in pups of both sexes from pregnant fed LPD. Moreover, the concentrations of two brain peptides neurotransmitters, thyrotropin-releasing hormone (TRH), and its biologically active metabolite, histidyl-proline diketopiperazine Cyclo (His-Pro), were elevated in the pups from LPD-fed mothers. In contrast, there was no significant difference in brain protein or brain peptides in pups from pair-fed mothers vs. pups from mothers fed ad libitum. These data suggest that qualitative alterations of the protein component in maternal dietary composition have deleterious effects upon the ontogeny of the rat fetal CNS, as reflected by reduced total protein and elevated concentrations of TRH and Cyclo (His-Pro).     

The effects of thyrotropin-releasing hormone on cyclic AMP accumulation in rabbit cerebral cortical tissue in the presence and absence of CNS depressants

The $\text{in vitro}$ effects of thyrotropin-releasing hormone on cAMP accumulation in cortical brain slices from rabbits is reported. Incubation of cortical tissue at three concentrations of thyrotropin-releasing hormone (1,2,5nM) had no discernible effects on baseline cAMP levels. When cortical tissue was incubated in the presence of pentobarbital (.5mM) or if cortical tissue was taken from animals pretreated with α-methyl-p-tyrosine (α-MPT), the baseline cAMP accumulation was depressed. This depression could be eliminated by the addition of TRH to the incubation media. Where cortical tissue from atropine-pretreated animals was used or when atropine was added to the incubation media, there was an increase in baseline cAMP accumulation which was unaffected by addition of TRH. These results show that TRH can modify cAMP accumulation in mammalian cortical brain tissue but this ability may only become evident in situations where normal cAMP concentration has been depressed.     

Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay

We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an ED50 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, beta-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 ng/mg tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.     

Zinc may play a role in the regulation of thyrotropin function

We studied the in vitro and in vivo influence of physiologically relevant zinc concentrations on the thyrotropin function both at the pituitary and hypothalamic level. Zinc gluconate (Zn Glu) concentrations from 5 to 100 microM decreased basal TSH release from anterior pituitary gland in vitro, but did not affect TSH-stimulated release by TRH, cAMP or high K+ concentrations. Zn Glu altered neither the basal nor stimulated production of TRH by hypothalami in vitro. In vivo brain third ventricle injection of Zn Glu decreased serum TSH 30-60 min after injection. The ability of physiological concentrations of zinc to influence TSH secretion both in vitro and in vivo suggest that this trace element might be involved in the regulation of thyrotropin function.     

Pyroglutamyl [correction of Pyroglutamil] -peptidase I activity in the cortex of the cat brain during development.

Thyrotropin Releasing Hormone (TRH) is a principal regulator of thyroid system function. However, significant concentrations of TRH were found throughout the central nervous system, the cortex being one of the areas most richly endowed with thyroliberin. Research concerning the functional role of this brain peptide is performed, in part, by studying peptidase enzymes which may be involved in the inactivation of the peptide. The pGlu-His bond is cleaved by two pyroGlu-peptidases: I (soluble) and II (membrane-bound). In the present investigation, developmental activity of the soluble form is described in the cortices of the cat brain. The selected maturation stages were 15 and 30 days postnatal. The cortices were the frontal, parietal, area 17 and areas 18 and 19 as a whole, distinguishing brain hemispheres in all cases. PyroGlu-aminopeptidase I activity increased significantly with age in all the brain regions except area 17. It is suggested that this enzyme activity plays a part in the neurochemical changes that take place during brain maturation.     

Effects of the dopamine agonist cabergoline on the pulsatile and TRH-induced secretion of prolactin, LH, and testosterone in male beagle dogs

In the present study, the pulsatile serum profiles of prolactin, LH and testosterone were investigated in eight clinically healthy fertile male beagles of one to six years of age. Serum hormone concentrations were determined in blood samples collected at 15 min intervals over a period of 6 h before (control) and six days before the end of a four weeks treatment with the dopamine agonist cabergoline (5 microg kg(-1) bodyweight/day). In addition, the effect of cabergoline administration was investigated on thyrotropin-releasing hormone (TRH)-induced changes in the serum concentrations of these hormones. In all eight dogs, the serum prolactin concentrations (mean 3.0 +/- 0.3 ng ml(-1)) were on a relatively constant level not showing any pulsatility, while the secretion patterns of LH and testosterone were characterised by several hormone pulses. Cabergoline administration caused a minor but significant reduction of the mean prolactin concentration (2.9 +/- 0.2 ng ml(-1), p < 0.05) and did not affect the secretion of LH (mean 4.6 +/- 1.3 ng ml(-1) versus 4.4 +/- 1.7 ng ml(-1)) or testosterone (2.5 +/- 0.9 ng ml(-1) versus 2.4 +/- 1.2 ng ml(-1)). Under control conditions, a significant prolactin release was induced by intravenous TRH administration (before TRH: 3.8 +/- 0.9 ng ml(-1), 20 min after TRH: 9.1 +/- 5.9 ng ml(-1)) demonstrating the role of TRH as potent prolactin releasing factor. This prolactin increase was almost completely suppressed under cabergoline medication (before TRH: 3.0 +/- 0.2 ng ml(-1), 20 min after TRH: 3.3 +/- 0.5 ng ml(-1)). The concentrations of LH and testosterone were not affected by TRH administration. The results of these studies suggest that dopamine agonists mainly affect suprabasal secretion of prolactin in the dog.