Sex-dependent and -independent regulation of thyrotropin-releasing hormone expression in the hypothalamic dorsomedial nucleus by negative energy balance, exercise, and chronic stress.

The dorsomedial nucleus of the hypothalamus (DMH) is part of the brain circuits that modulate organism responses to the circadian cycle, energy balance, and psychological stress. A large group of thyrotropin-releasing hormone (Trh) neurons is localized in the DMH; they comprise about one third of the DMH neurons that project to the lateral hypothalamus area (LH). We tested their response to various paradigms. In male Wistar rats, food restriction during adulthood, or chronic variable stress (CVS) during adolescence down-regulated adult DMH Trh mRNA levels compared to those in sedentary animals fed ad libitum; two weeks of voluntary wheel running during adulthood enhanced DMH Trh mRNA levels compared to pair-fed rats. Except for their magnitude, female responses to exercise were like those in male rats; in contrast, in female rats CVS did not change DMH Trh mRNA levels. A very strong negative correlation between DMH Trh mRNA levels and serum corticosterone concentration in rats of either sex was lost in CVS rats. CVS canceled the response to food restriction, but not that to exercise in either sex. TRH receptor 1 (Trhr) cells were numerous along the rostro-caudal extent of the medial LH. In either sex, fasting during adulthood reduced DMH Trh mRNA levels, and increased LH Trhr mRNA levels, suggesting fasting may inhibit the activity of TRHDMH->LH neurons. Thus, in Wistar rats DMH Trh mRNA levels are regulated by negative energy balance, exercise and chronic variable stress through sex-dependent and -independent pathways.

Evolution of thyrotropin-releasing factor extracellular communication units.

Thyroid hormones (THs) are ancient signaling molecules that contribute to the regulation of metabolism, energy homeostasis and growth. In vertebrates, the hypothalamus-pituitary-thyroid (HPT) axis links the corresponding organs through hormonal signals, including thyrotropin releasing factor (TRF), and thyroid stimulating hormone (TSH) that ultimately activates the synthesis and secretion of THs from the thyroid gland. Although this axis is conserved among most vertebrates, the identity of the hypothalamic TRF that positively regulates TSH synthesis and secretion varies. We review the evolution of the hypothalamic factors that induce TSH secretion, including thyrotropin-releasing hormone (TRH), corticotrophin-releasing hormone (CRH), urotensin-1-3, and sauvagine, and non-mammalian glucagon-like peptide in metazoans. Each of these peptides is part of an extracellular communication unit likely composed of at least 3 elements: the peptide, G-protein coupled receptor and bioavailability regulator, set up on the central neuroendocrine articulation. The bioavailability regulators include a TRH-specific ecto-peptidase, pyroglutamyl peptidase II, and a CRH-binding protein, that together with peptide secretion/transport rate and transduction coupling and efficiency at receptor level shape TRF signal intensity and duration. These vertebrate TRF communication units were coopted from bilaterian ancestors. The bona fide elements appeared early in chordates, and are either used alternatively, in parallel, or sequentially, in different vertebrate classes to control centrally the activity of the HPT axis. Available data also suggest coincidence between apparition of ligand and bioavailability regulator.

A screen for modulators reveals that orexin-A rapidly stimulates thyrotropin releasing hormone expression and release in hypothalamic cell culture.

In the paraventricular nucleus of the mammalian hypothalamus, hypophysiotropic thyrotropin releasing hormone (TRH) neurons integrate metabolic information and control the activity of the thyroid axis. Additional populations of TRH neurons reside in various hypothalamic areas, with poorly defined connections and functions, albeit there is evidence that some may be related to energy balance. To establish extracellular modulators of TRH hypothalamic neurons activity, we performed a screen of neurotransmitters effects in hypothalamic cultures. Cell culture conditions were chosen to facilitate the full differentiation of the TRH neurons; these conditions had permitted the characterization of the effects of known modulators of hypophysiotropic TRH neurons. The major end-point of the screen was Trh mRNA levels, since they are generally rapidly (0.5-3h) modified by synaptic inputs onto TRH neurons; in some experiments, TRH cell content or release was also analyzed. Various modulators, including histamine, serotonin, ß-endorphin, met-enkephalin, and melanin concentrating hormone, had no effect. Glutamate, as well as ionotropic agonists (kainate and N-Methyl-d-aspartic acid), increased Trh mRNA levels. Baclofen, a GABAB receptor agonist, and dopamine enhanced Trh mRNA levels. An endocannabinoid receptor 1 inverse agonist promoted TRH release. Somatostatin increased Trh mRNA levels and TRH cell content. Orexin-A rapidly increased Trh mRNA levels, TRH cell content and release, while orexin-B decreased Trh mRNA levels. These data reveal unaccounted regulators, which exert potent effects on hypothalamic TRH neurons in vitro.

Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

PURPOSE: Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. METHODS: Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. RESULTS: Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. CONCLUSIONS: Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

Advances in TRH signaling.

The activity of the hypothalamus-pituitary-thyroid axis (HPT) is coordinated by hypophysiotropic thyrotropin releasing hormone (TRH) neurons present in the paraventricular nucleus of the hypothalamus. Hypophysiotropic TRH neurons act as energy sensors. TRH controls the synthesis and release of thyrotropin, which activates the synthesis and secretion of thyroid hormones; in target tissues, transporters and deiodinases control their local availability. Thyroid hormones regulate many functions, including energy homeostasis. This review discusses recent evidence that covers several aspects of TRH role in HPT axis regulation. Knowledge about the mechanisms of TRH signaling has steadily increased. New transcription factors engaged in TRH gene expression have been identified, and advances made on how they interact with signaling pathways and define the dynamics of TRH neurons response to acute and/or long-term influences. Albeit yet incomplete, the relationship of TRH neurons activity with positive energy balance has emerged. The importance of tanycytes as a central relay for the feedback control of the axis, as well as for HPT responses to alterations in energy balance, and other stimuli has been reinforced. Finally, some studies have started to shed light on the interference of prenatal and postnatal stress and nutrition on HPT axis programing, which have confirmed the axis susceptibility to early insults.

Neonatal Maternal Separation Alters, in a Sex-Specific Manner, the Expression of TRH, of TRH-Degrading Ectoenzyme in the Rat Hypothalamus, and the Response of the Thyroid Axis to Starvation.

Hypothalamic-pituitary-thyroid (HPT) axis activity is important for energy homeostasis, and is modified by stress. Maternal separation (MS) alters the stress response and predisposes to metabolic disturbances in the adult. We therefore studied the effect of MS on adult HPT axis activity. Wistar male and female pups were separated from their mothers 3 h/d during postnatal day (PND)2-PND21 (MS), or left nonhandled (NH). Open field and elevated plus maze tests revealed increased locomotion in MS males and anxiety-like behavior in MS females. At PND90, MS females had increased body weight gain, Trh expression in the hypothalamic paraventricular nucleus, and white adipose tissue mass. MS males had increased expression of TRH-degrading enzyme in tanycytes, reduced TSH and T3, and enhanced corticosterone serum concentrations. MS stimulated brown adipose tissue deiodinase 2 activity in either sex. Forty-eight hours of fasting (PND60) augmented serum corticosterone levels similarly in MS or NH females but more in MS than in NH male rats. MS reduced the fasting-induced drop in hypothalamic paraventricular nucleus-Trh expression of males but not of females and abolished the fasting-induced increase in Trh expression in both sexes. Fasting reduced serum concentrations of TSH, T4, and T3, less in MS than in NH males, whereas in females, TSH decreased in MS but not in NH rats, but T4 and T3 decreased similarly in NH and MS rats. In conclusion, MS produced long-term changes in the activity of the HPT axis that were sex specific; response to fasting was partially blunted in males, which could affect their adaptive response to negative energy balance.

60 YEARS OF NEUROENDOCRINOLOGY: TRH, the first hypophysiotropic releasing hormone isolated: control of the pituitary-thyroid axis.

This review presents the findings that led to the discovery of TRH and the understanding of the central mechanisms which control hypothalamus-pituitary-thyroid axis (HPT) activity. The earliest studies on thyroid physiology are now dated a century ago when basal metabolic rate was associated with thyroid status. It took over 50 years to identify the key elements involved in the HPT axis. Thyroid hormones (TH: T4 and T3) were characterized first, followed by the semi-purification of TSH whose later characterization paralleled that of TRH. Studies on the effects of TH became possible with the availability of synthetic hormones. DNA recombinant techniques facilitated the identification of all the elements involved in the HPT axis, including their mode of regulation. Hypophysiotropic TRH neurons, which control the pituitary-thyroid axis, were identified among other hypothalamic neurons which express TRH. Three different deiodinases were recognized in various tissues, as well as their involvement in cell-specific modulation of T3 concentration. The role of tanycytes in setting TRH levels due to the activity of deiodinase type 2 and the TRH-degrading ectoenzyme was unraveled. TH-feedback effects occur at different levels, including TRH and TSH synthesis and release, deiodinase activity, pituitary TRH-receptor and TRH degradation. The activity of TRH neurons is regulated by nutritional status through neurons of the arcuate nucleus, which sense metabolic signals such as circulating leptin levels. Trh expression and the HPT axis are activated by energy demanding situations, such as cold and exercise, whereas it is inhibited by negative energy balance situations such as fasting, inflammation or chronic stress. New approaches are being used to understand the activity of TRHergic neurons within metabolic circuits.

Fasting Enhances Pyroglutamyl Peptidase II Activity in Tanycytes of the Mediobasal Hypothalamus of Male Adult Rats.

Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of ß2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN.

TGFß2 regulates hypothalamic Trh expression through the TGFß inducible early gene-1 (TIEG1) during fetal development.

The hypothalamus regulates the homeostasis of the organism by controlling hormone secretion from the pituitary. The molecular mechanisms that regulate the differentiation of the hypothalamic thyrotropin-releasing hormone (TRH) phenotype are poorly understood. We have previously shown that Klf10 or TGFß inducible early gene-1 (TIEG1) is enriched in fetal hypothalamic TRH neurons. Here, we show that expression of TGFß isoforms (1-3) and both TGFß receptors (TßRI and II) occurs in the hypothalamus concomitantly with the establishment of TRH neurons during late embryonic development. TGFß2 induces Trh expression via a TIEG1 dependent mechanism. TIEG1 regulates Trh expression through an evolutionary conserved GC rich sequence on the Trh promoter. Finally, in mice deficient in TIEG1, Trh expression is lower than in wild type animals at embryonic day 17. These results indicate that TGFß signaling, through the upregulation of TIEG1, plays an important role in the establishment of Trh expression in the embryonic hypothalamus.

Transcriptional profiling of fetal hypothalamic TRH neurons.

BACKGROUND: During murine hypothalamic development, different neuroendocrine cell phenotypes are generated in overlapping periods; this suggests that cell-type specific developmental programs operate to achieve complete maturation. A balance between programs that include cell proliferation, cell cycle withdrawal as well as epigenetic regulation of gene expression characterizes neurogenesis. Thyrotropin releasing hormone (TRH) is a peptide that regulates energy homeostasis and autonomic responses. To better understand the molecular mechanisms underlying TRH neuron development, we performed a genome wide study of its transcriptome during fetal hypothalamic development. RESULTS: In primary cultures, TRH cells constitute 2% of the total fetal hypothalamic cell population. To purify these cells, we took advantage of the fact that the segment spanning -774 to +84 bp of the Trh gene regulatory region confers specific expression of the green fluorescent protein (GFP) in the TRH cells. Transfected TRH cells were purified by fluorescence activated cell sorting, various cell preparations pooled, and their transcriptome compared to that of GFP- hypothalamic cells. TRH cells undergoing the terminal phase of differentiation, expressed genes implicated in protein biosynthesis, intracellular signaling and transcriptional control. Among the transcription-associated transcripts, we identified the transcription factors Klf4, Klf10 and Atf3, which were previously uncharacterized within the hypothalamus. CONCLUSION: To our knowledge, this is one of the first reports identifying transcripts with a potentially important role during the development of a specific hypothalamic neuronal phenotype. This genome-scale study forms a rational foundation for identifying genes that might participate in the development and function of hypothalamic TRH neurons.

The Krüppel-like factor 4 controls biosynthesis of thyrotropin-releasing hormone during hypothalamus development.

Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Krüppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4(-/-) mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4(-/-) mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons.

T3 differentially regulates TRH expression in developing hypothalamic neurons in vitro.

Triiodothyronine (T3) plays an important role during development of the central nervous system. T3 effects on gene expression are determined in part by the type of thyroid hormone receptors (TRs) expressed in a given cell type. Previous studies have demonstrated that thyrotropin releasing hormone (TRH) transcription in the adult hypothalamus is subjected to negative regulation by thyroid hormones. However, the role of T3 on the development of TRH expression is unknown. In this study we used primary cultures derived from 17-day-old fetal rat hypothalamus to analyze the effects of T3 on TRH gene expression during development. T3 increased TRH mRNA expression in immature cultures, but decreased it in mature cultures. In addition, T3 up-regulated TRalpha1 and TRbeta2 mRNA expression. TRalpha1 expression coincided chronologically with that of TRH in the rat hypothalamus in vivo. Maturation of TRH expression in the hypothalamus may involve T3 acting through TRalpha1.

The PKC and ERK/MAPK pathways regulate glucocorticoid action on TRH transcription.

Biosynthesis of TRH, a neuropeptide involved in energy homeostasis, is modulated by glucocorticoids. TRH mRNA and peptide levels are increased upon incubation of hypothalamic cells with dexamethasone or with cAMP analogs but when combined, a mutual antagonism is observed. These effects are observed at the transcriptional level and on binding of glucocorticoid receptor (GR) or pCREB to the composite GRE (cGRE) and CRE-2 sites of TRH promoter. The present work studied the involvement of PKC and MAPK pathways on the effect of dexamethasone and on its interaction with cAMP signaling in hypothalamic cell cultures. PKC or MEK inhibition abolished dexamethasone-stimulatory effect on TRH mRNA levels, as well as its interference with the stimulatory effect of 8Br-cAMP. Binding of nuclear extracts from hypothalamic or neuroblastoma cells stimulated with dexamethasone or 8Br-cAMP to oligonucleotides containing the CRE or cGRE sites of TRH gene promoter was decreased if cells were preincubated with PKC or MEK inhibitors. Mutations on the AP-1 or the GRE half sites of cGRE showed that GR binds as an heterodimer on cGRE, and PKC or MEK inhibitors diminish binding at the AP-1 site. PKC and ERK signaling thus modulate GR activity and its interaction with CREB or AP-1 at the TRH gene promoter.

BDNF up-regulates pre-pro-TRH mRNA expression in the fetal/neonatal paraventricular nucleus of the hypothalamus. Properties of the transduction pathway.

Brain derived neurotrophic factor (BDNF) increases the levels of pre-pro-thyrotropin releasing hormone (TRH) mRNA in fetal rodent hypothalamic neurons that express TrkB receptors. The present studies aimed at better understanding the role of BDNF in establishing and maintaining the TRH phenotype in hypothalamic neurons during early development. To determine where BDNF regulates the expression of pre-pro-TRH mRNA in vivo, we compared the hypothalamic distribution of pre-pro-TRH mRNA to that of TrkB mRNA. Full-length TrkB (FL-TrkB) mRNA was detected earlier in development than pre-pro-TRH mRNA in the region that gives rise to the paraventricular nucleus of the hypothalamus (PVN). We also evaluated the effects of BDNF on the expression of pre-pro-TRH mRNA in vitro. BDNF up-regulated the levels of pre-pro-TRH mRNA in primary cell cultures obtained from the hypothalamus or the PVN of 17 days old fetuses or newborn rats. This effect was abolished by PD98059, an inhibitor of the mitogen-activated protein kinase kinase (MEK) 1/2 or 5. The effect of BDNF on pre-pro-TRH mRNA levels was reversible. The continuous application of BDNF led to a desensitization of the response at day 10 in vitro, an effect that correlated with a drop in the levels of FL-TrkB protein. In conclusion, BDNF enhances the expression of pre-pro-TRH mRNA in PVN neurons. This effect is reversible, decreases with time, and requires an active MEK. BDNF may contribute to the enhancement of pre-pro-TRH mRNA expression in the hypothalamic PVN during development.

Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain.

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

An improved method for the expression of TRH in serum-supplemented primary cultures of fetal hypothalamic cells.

Primary cultures of dispersed cells from fetal nervous tissue are extensively used for studying multiple neuronal properties. Analyses of the developmental expression of thyrotropin releasing hormone (TRH; pglu-his-pro-NH(2)) biosynthesis in primary cultures of fetal dissociated hypothalamic cells have shown that cellular TRH levels per dish increase with time in culture, after a lag period of a few days, but do not attain the values observed in vivo, hampering its use as a model system for the study of peptide biosynthesis and release. We have demonstrated that homologous conditioned medium (CM) enhances TRH expression in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. We report here experimental conditions that allow the expression, during the second or third week in vitro, of higher cellular TRH levels than previously described in primary cultures of dissociated hypothalamic cells from 17-day rat fetuses. The medium used was Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), vitamins, glucose, glutamine and insulin (DMEM-S). Cellular levels of TRH/mg protein increased with cell density between 1 and 2.7x10(6) cells per 35-mm dish. Addition of 10(-5) M cytosine arabinoside (CAr) at the 4th day in vitro (DIV) improved TRH cell content per dish compared to addition at 5 DIV; 2.5-5x10(-5) M bromodeoxyuridine added at seeding reduced cell survival and did not enhance TRH levels, in comparison to CAr-treated cultures. Addition of ascorbic acid (0.5-1x10(-4) M) increased TRH levels per dish. Substitution of DMEM by DMEM-F12 (1:1) did not improve TRH levels. Cellular levels of TRH, in Neurobasal plus B27 (a serum-free medium), were similar to levels in serum-supplemented media. In the optimized conditions, a small number of pro-TRH mRNA expressing cells (2% of total cells) was detected by in situ hybridization; 40% coexpressed the pro-protein convertase PC1 mRNA. Conditioning the medium, controlling glial proliferation, and adding ascorbic acid improved the expression of TRH in primary culture of hypothalamic cells in DMEM-S.