Immunohistochemical localization in the rat brain of the precursor for thyrotropin-releasing hormone.

A rabbit antiserum to a peptide sequence present in the precursor for thyrotropin-releasing hormone (proTRH), deduced from cloned amphibian-skin complementary DNA, was raised by immunization with the synthetic decapeptide Cys-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Cys (proTRH-SH). Immunohistochemical studies on rat brain tissue showed staining of neuronal perikarya in the parvicellular division of the paraventricular nucleus of the hypothalamus and the raphe complex of the medulla, identical to that already described for thyrotropin-releasing hormone (TRH). Immunostaining was abolished by preincubation with proTRH-SH (10(-6)M) but not TRH (10(-5)M). Both TRH precursor and TRH were located in neurons of the paraventricular nucleus. However, in contrast to the findings for TRH, no staining was observed in axon terminals of the median eminence. These results suggest that a TRH precursor analogous to that reported in frog skin is present in the rat brain and that TRH in the mammalian central nervous system is a product of ribosomal biosynthesis.

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.

Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus.

Thyroid hormone is important in the regulation of synthesis and secretion of thyroid-stimulating hormone (TSH) in the anterior pituitary, but its role in the control of hypothalamic thyrotropin-releasing hormone (TRH) is controversial. To determine whether thyroid hormone regulates the function of TRH in the hypothalamic tuberoinfundibular system, a study was made of the effect of hypothyroidism on thyrotropin-releasing hormone messenger RNA (proTRH mRNA) and TRH prohormone in the rat paraventricular nucleus. Extracts of rat hypothalamic paraventricular nucleus were examined by quantitative Northern blot analysis, and coronal sections of rat brain were examined by in situ hybridization histochemistry and immunocytochemistry. A nearly twofold increase in proTRH mRNA was observed in hypothyroid animals; this increase could be obliterated by levothyroxine treatment, suggesting an inverse relation between circulating thyroid hormone and proTRH mRNA. In situ hybridization showed that this response occurred exclusively in medial parvocellular neurons of the paraventricular nucleus. A simultaneous increase in proTRH mRNA and immunoreactive TRH prohormone in this region suggests that hypothyroidism induces both transcription and translation of the TRH prohormone in the paraventricular nucleus.

Thyrotropin-releasing hormone precursor: characterization in rat brain.

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity.

Brain-derived neurotrophic factor has been associated previously with the regulation of food intake. To help elucidate the role of this neurotrophin in weight regulation, we have generated conditional mutants in which brain-derived neurotrophic factor has been eliminated from the brain after birth through the use of the cre-loxP recombination system. Brain-derived neurotrophic factor conditional mutants were hyperactive after exposure to stressors and had higher levels of anxiety when evaluated in the light/dark exploration test. They also had mature onset obesity characterized by a dramatic 80-150% increase in body weight, increased linear growth, and elevated serum levels of leptin, insulin, glucose, and cholesterol. In addition, the mutants had an abnormal starvation response and elevated basal levels of POMC, an anorexigenic factor and the precursor for alpha-MSH. Our results demonstrate that brain derived neurotrophic factor has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.

Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Gene expression for agouti-related protein (AGRP), an endogenous antagonist of melanocortin receptors, has been localized to the hypothalamic arcuate nucleus, where it colocalizes with neuropeptide Y (NPY). Having reported that the NPY innervation of hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) originates primarily from NPY-producing neurons in the arcuate nucleus, here we examined the possibility that TRH neurons in the PVN are similarly innervated by AGRP nerve terminals. Using immunohistochemistry, AGRP-containing cell bodies were found almost exclusively in the arcuate nucleus, but their projections were distributed widely in the hypothalamus, most conspicuously in the paraventricular (PVN), arcuate and dorsomedial nuclei, and the posterior hypothalamic area. Ablation of the arcuate nucleus by the neonatal administration of monosodium glutamate obliterated nearly all AGRP-immunoreactivity in the hypothalamus. In the PVN, double-labeling light and electron microscopic immunohistochemistry revealed that TRH neurons receive dense innervation by AGRP nerve terminals, with the frequent occurrence of axosomatic and axodendritic synapses (mainly of the symmetrical type). These findings provide morphological basis to hypothesize a role for AGRP in the arcuato-paraventricular pathway, in the down-regulation of the hypothalamic-pituitary-thyroid axis, which occurs as an adaptive response to starvation.

Immunohistochemical localization of thyrotropin-releasing hormone in the rat hypothalamus and pituitary.

The distribution of immunoreactive TRH in the rat hypothalamus and pituitary was demonstrated using the peroxidase-antiperoxidase technique after rapid fixation of the rat brain with 5% acrolein. Widespread reaction product was identified in neuronal processes throughout the hypothalamus, with dense labeling in the median eminence, dorsomedial nucleus, parvocellular division of the paraventricular nucleus, perifornical region, periventricular nucleus, and organum vasculosum of the lamina terminalis. A striking accumulation of immunoreactive TRH was also noted throughout the posterior pituitary, where fibers appeared to terminate in grape-like swellings. Peroxidase-positive perikarya were best seen after colchicine pretreatment and were distributed in many regions of the hypothalamus. The greatest density of immunoreactive neurons was in the suprachiasmatic preoptic nucleus, parvocellular subdivision of the paraventricular nucleus, perifornical region, dorsomedial nucleus, and baso-lateral hypothalamus. These data are consistent with the role of TRH as a hypophysiotropic hormone, a regulator of the posterior pituitary, and a neurotransmitter or neuromodulator of neurons in other regions of the hypothalamus.

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.

Changes in adrenal status affect hypothalamic thyrotropin-releasing hormone gene expression in parallel with corticotropin-releasing hormone.

Glucocorticoids are well known to influence the secretion of TSH from the anterior pituitary gland, although it is uncertain whether its site of action is on the hypothalamus, pituitary, or both. To determine whether glucocorticoids can modulate the concentration of pro-TRH gene expression in hypothalamic hypophysiotropic neurons, we measured the content of pro-TRH messenger RNA (mRNA) in the paraventricular nucleus (PVN) of adrenalectomized and corticosterone- and dexamethasone-treated rats compared to that in control populations using in situ hybridization histochemistry. Adrenalectomy resulted in the expected increase in corticotropin-releasing hormone mRNA in the PVN and was accompanied by a parallel rise in pro-TRH mRNA (68.3%; P < 0.05). Conversely, corticosterone and dexamethasone both resulted in profound reduction in corticotropin-releasing hormone mRNA in the PVN and a parallel reduction in pro-TRH mRNA (43.2% and 73.2% respectively; P < 0.05). No significant differences were observed in pro-TRH mRNA in the lateral hypothalamus in any of the groups. These data suggest that glucocorticoids can influence the concentration of pro-TRH mRNA in a cell-specific manner and thereby could result in changes in the biosynthesis and release of TRH in hypophysiotropic neurons of the PVN.

Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system.

The distribution of immunoreactive TRH prohormone in the rat central nervous system was studied by immunocytochemistry using an antiserum raised against a synthetic decapeptide hypothesized to represent a portion of the mammalian TRH precursor protein. Reaction product was identified in several regions of the brain in a distribution typical of that previously described for the tripeptide. In contrast to TRH, however, immunoreactive pro-TRH was largely confined to neuronal perikarya and only rarely seen in axons or axon terminals. In addition, immunoreactive pro-TRH was present in portions of the telencephalon and brainstem where TRH has not previously been described in neurons by immunocytochemistry. These studies indicate that in most regions of the brain the TRH prohormone is rapidly processed within the cell soma and not during axonal transport, and raise the possibility that in certain regions of the brain processing of the prohormone may be to non-TRH peptides, which may be of biological importance.

Immunocytochemical distribution in rat brain of putative peptides derived from thyrotropin-releasing hormone prohormone.

Processing of the TRH prohormone (Pro-TRH), a protein of approximately 26,000 mol wt, could yield 5 copies of TRH, as well as extended forms of TRH and several other non-TRH peptides. To determine whether some of these peptides are formed and transported by axons in the rat brain, we used antiserum to synthetic peptides corresponding to portions of pro-TRH. These included the N-tyrosyl analogs [Tyr0]prepro-TRH-(25-50) (pYE27) and [Tyr1]prepro-TRH-(53-74) (pYT22) contained within the N-terminal flanking region of the prohormone, the N-tyrosyl analog [Tyr0]prepro-TRH-(165-186) (pYS23), expanding the fourth progenitor sequence of TRH in the midportion of the prohormone, and the synthetic peptide pAC12 corresponding to the first 12 amino acids of the C-terminal flanking region or prepro-TRH-(208-219). All antisera showed staining in neuronal perikarya and processes in all regions of the brain previously demonstrated to immunostain for TRH, including dense innervation of the external zone of the median eminence. In addition, these antisera immunostained regions of the brain not previously immunopositive for TRH. Not all regions reactive with antiserum to [Tyr0]prepro-TRH-(25-50) were also recognized by anti-pYT, -pYS, and -pAC. These studies confirm the presence of the deduced non-TRH sequences within the TRH precursor and their formation and transport in vivo in the central nervous system. The presence of immunoreactivity in regions of the brain that do not contain TRH and the variability of immunostaining of the different antisera in some of these regions suggest regional preferential processing of pro-TRH to other peptides that may be biologically active.

Thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is dependent upon feedback regulation by both triiodothyronine and thyroxine.

The biosynthesis of TRH in hypophysiotropic neurons of the paraventricular nucleus (PVN) is inversely regulated by feedback effects of circulating levels of thyroid hormones. As the PVN contains little or no deiodinase activity, the enzyme necessary to convert T4 to biologically active T3, we determined whether feedback inhibition of pro-TRH mRNA in thyroid hormone-sensitive neurons of the PVN is mediated exclusively by circulating levels of T3. The concentration of pro-TRH mRNA in the PVN of hypothyroid male rats receiving constant infusions of T3 over 7 days from ip implanted osmotic minipumps was studied by in situ hybridization histochemistry using computerized image analysis. Pro-TRH mRNA could not be suppressed to euthyroid levels by an infusion of T3 that returned plasma T3 levels to normal and required the infusion of higher concentrations of T3 that elevated plasma T3 into the supranormal range. By regression analysis, the mean concentration of plasma T3 required to suppress pro-TRH mRNA to euthyroid levels was estimated to be 110.3 ng/dl, similar to the amount of T3 estimated to be necessary to suppress TSH secretion from the anterior pituitary (108.7 ng/dl). We conclude that both T3 and T4 contribute to feedback inhibition of TRH biosynthesis in hypophysiotropic neurons of the PVN and propose that the effects of T4 on the PVN could be mediated after its monodeiodination at a different locus within the brain.

Identification and characterization of thyrotropin-releasing hormone precursor peptides in rat brain.

The sequence of rat hypothalamic pro-TRH, deduced by sequencing of cDNA, contains five copies of the TRH progenitor sequence Gln-His-Pro-Gly flanked by paired basic amino acid sequences. The TRH prohormone also contains leader and trailer sequences and four intervening sequences. We have developed two RIAs against synthetic peptides corresponding to sequences within the deduced pro-TRH sequence and have used these assays to identify and partially characterize four pro-TRH-derived peptides distinct from TRH in extracts of rat brain tissue. Two of these peptides contain incompletely processed TRH sequences; the other two peptides are probably derived from the N-terminal leader sequence. The presence of these authentic pro-TRH-derived peptides indicates that pro-TRH may give rise to a family of peptides other than TRH, some of which may be of biological significance.

Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus.

The association of neuropeptide-Y (NPY)-immunoreactive (IR) axon terminals with TRH-synthesizing neurons in the rat hypothalamic paraventricular nucleus (PVN) has been studied. Immunocytochemical single and double labeling studies were performed at both light and electron microscopic levels using antiserum to NPY and, as a marker of TRH-containing neurons, antisera recognizing the N-terminal flanking peptides of the TRH prohormone, prepro-TRH-(25-50) and prepro-TRH-(53-74). At the light microscopic level, a diffuse group of TRH-IR cell bodies were observed in the anterior parvocellular subdivision of the PVN and became more numerous and densely clustered in the medial and periventricular parvocellular subdivisions. NPY-IR fibers were observed to innervate all subdivisions of the PVN, but were particularly dense in the anterior, medial, and periventricular parvocellular subdivisions of the nucleus, where they appeared to contact TRH-synthesizing perikarya and neuronal processes. At the ultrastructural level, numerous NPY-IR axon terminals containing labeled vesicles were either tightly juxtaposed to TRH-producing neurons or seen to establish both symmetric and asymmetric synaptic contacts with TRH-containing cell bodies and dendrites. Some NPY-IR axon terminals also established synaptic contacts with unlabeled PVN perikarya and processes or were found in close apposition to blood vessels. These data provide a morphological basis to suggest NPY-mediated neuroendocrine regulation over the biosynthesis and/or secretion of TRH in the PVN. Reports of the colocalization of NPY and catecholamines in the same axon terminals raises the possibility of a potential interaction between NPY and catecholamines to influence TRH neurons in the PVN. Morphological evidence for synaptic interactions between NPY-IR axon terminals and non-TRH-containing neurons in the PVN further suggests that this peptide may influence other neuroendocrine systems.

Endotoxin-induced corticotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin-1.

In the acute phase of bacterial infection, a variety of cytokines, including interleukin-1 (IL-1), are elicited by bacterial endotoxin in both the periphery and the central nervous system. Bacterial endotoxin has been previously reported to profoundly activate the hypothalamic-pituitary-adrenal axis, resulting in elevated glucocorticoid secretion that may serve an important role as part of the inhibitory feedback mechanisms on the activated immune system. To determine whether IL-1 acts within the brain to mediate endotoxin-induced CRH gene expression in the hypothalamic paraventricular nucleus (PVN), we studied the effect of administering the human IL-1 receptor antagonist (IL-1ra) into the brain, a competitive inhibitor of IL-1, on CRH gene expression in the PVN after systemic lipopolysaccharide (LPS) treatment. Eight hours after the ip administration of LPS, the paraventricular CRH mRNA content was elevated 3-to 4-fold (P < 0.01) compared to the control value, and this elevation could be completely abolished by central IL-1ra pretreatment (P < 0.05 compared to LPS-treated group; P > 0.05 compared to controls). In contrast, systemic IL-1ra administration did not inhibit endotoxin-induced CRH gene expression in the PVN. These studies demonstrate that LPS stimulates hypothalamic CRH by a mechanism that involves the action of IL-1 within the central nervous system and may proceed independently of peripheral actions of IL-1 circulating in the bloodstream.

Expression of thyroid hormone receptor beta 2 in rat hypothalamus.

A polymerase chain reaction based assay was used to evaluate expression of thyroid hormone receptor beta 2 mRNA in rat hypothalamus. Expression was detected in the arcuate, ventromedial and paraventricular nuclei, as well as the median eminence. Trace expression was found in the dorsomedial nucleus, but no expression of thyroid hormone receptor beta 2 was detected in the lateral hypothalamus or the preoptic region. The results indicate that, contrary to previous belief, expression of thyroid hormone receptor beta 2 is not confined to the anterior pituitary.

Hypothyroidism increases vasoactive intestinal polypeptide (VIP) immunoreactivity and gene expression in the rat hypothalamic paraventricular nucleus.

Vasoactive intestinal polypeptide (VIP) is produced by neurons in the rat hypothalamic paraventricular nucleus (PVN) and may have an important role as a prolactin-releasing factor. Recent work from our laboratories has shown that thyroid hormone regulates the content of VIP and VIP mRNA in the rat anterior pituitary, but its effect on VIP in the PVN is not known. To determine whether thyroid hormone alters VIP biosynthesis in the PVN, we studied the effect of hypothyroidism on the content of immunoreactive (IR)-VIP and VIP mRNA in PVN neurons using histochemical techniques. By immunocytochemistry, only scattered IR-VIP fibers were present in the PVN of control animals whereas IR-VIP perikarya and fibers were present in hypothyroid rats. By in situ hybridization histochemistry, no labeled neurons were recognized in the PVN in control animals whereas PVN neurons were labeled in hypothyroid rats. These findings raise the possibility that hypothyroidism exerts negative feedback regulation on VIP-producing neurons in the PVN and suggest that this may be important to modulate the stimulatory effects of VIP on anterior and/or posterior pituitary function.

Distribution of immunoreactive human growth hormone-like material and thyrotropin-releasing hormone in the rat central nervous system: evidence for their coexistence in the same neurons.

The distribution of a substance with human GH (hCG)-like immunoreactivity was studied in the rat brain after rapid fixation with acrolein. Using this method of tissue preparation, the hGH-like material (hGH-LM) was found in several hypothalamic and extrahypothalamic regions of the brain and extended into the spinal cord and posterior pituitary. On serial sections, the distribution of the hGH-LM was observed to be identical to that of TRH throughout the neuraxis. Sequential immunostaining of the hGH-LM and TRH in the same tissue section revealed the coexistence of these two peptides in the same neuronal cell bodies in the hypothalamus and brain stem and in beaded processes in all regions of the central nervous system studied. These findings demonstrate the intimate association between the hGH-LM and TRH in the central nervous system and raise the possibility that the hGH-LM forms part of a precursor hormone from which TRH is derived.

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.