Development of pro-TRH gene expression in primary cultures of fetal hypothalamic cells

Developmental Brain Research 130 (2001) 73–81 www.elsevier.com / locate / bres

Research report

Development of pro-TRH gene expression in primary cultures of fetal hypothalamic cells ´ ´ Leonor Perez-Martınez, Jean-Louis Charli, Patricia Joseph-Bravo* ´ ´ Molecular, Instituto de Biotecnologıa ´ , Universidad Nacional Autonoma ´ ´ de Mexico , A.P. 510 -3, Cuernavaca, Departamento de Genetica y Fisiologıa Mor. 62271, Mexico Accepted 12 June 2001

Abstract Little is known about the temporal relationship and the sequential steps for peptide biosynthesis during the terminal differentiation of the peptide phenotype in central nervous system. Analysis of the TRH phenotype in primary cultures of rat fetal day 17 hypothalamic cells has shown that TRH levels start increasing only after a week in culture, in contrast with in vivo data showing a steady increase during late fetal life. The purpose of this study was to compare the developmental patterns of TRH and pro-TRH mRNA levels in vitro to determine whether the initial low and steady levels of TRH are due to deficient transcription. Pro-TRH mRNA levels were detected by semi-quantitative RT-PCR through the development of primary cultures of serum-supplemented hypothalamic fetal cells from 17 day old embryos. Pro-TRH mRNA levels per dish increased steadily since the beginning of the culture. In contrast, TRH levels per dish were low and stable during the first week increasing afterwards, but remaining low compared to equivalent in vivo values. Pro-TRH mRNA levels per hypothalamus increased between fetal day 17 and postnatal 14, suggesting that the in vitro pattern of pro-TRH mRNA development mimics that occurring in vivo. These data show that pro-TRH gene expression does not limit TRH accumulation in vitro suggesting that the transcriptional and post-transcriptional programs leading to peptide accumulation are established independently.  2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitters systems and channels Keywords: TRH; Hypothalamus; Primary culture; TRH biosynthesis; Pro-TRH

1. Introduction Neuropeptide biosynthesis is a sequential process involving multiple steps including transcription, translation, transport through the secretory pathway and processing of the precursor. External clues for the post-mitotic development of the capacity of central neurons to express neuropeptides are slowly emerging [6]. However, little is known about the temporal relationship between initiation of peptide precursor mRNA transcription and accumulation of peptide during terminal differentiation. Various populations of hypothalamic neurons synthesize the tripeptide thyrotropin releasing hormone (pglu-hisproNH 2 ; TRH). Pro-TRH mRNA and protein and TRH expressions are developmentally regulated in vivo. In the *Corresponding author. Tel.: 152-73-170-805; fax: 152-73-172-388. E-mail address: [email protected] (P. Joseph-Bravo).

rat diencephalon, cells expressing pro-TRH mRNA are first detected in lateral hypothalamus at fetal day 14, in ventromedial nucleus at day 15, in paraventricular nucleus (PVN) at day 16 and in preoptic area at day 17. In these nuclei, labeling intensity and number of TRH neurons increase during pre- and postnatal periods [5]. This is accompanied by an increase of pro-TRH mRNA levels [9] as well as an initial increase of pro-TRH protein levels [31]. These events are paralleled by increasing levels of various enzymes (and / or of their mRNAs) that process pro-TRH during pre and post-natal periods, including proprotein convertases PC1 and PC2, peptidyl a-amidating monooxygenase (PAM) and carboxypeptidase H [11,32,43]. TRH levels per hypothalamus increase steadily in vivo before and after birth [10,16,20,28]. Between embryonic day 22 (E22) and postnatal day 7 (P7), the subcellular distribution of TRH switches from its predominant association with small granules to an equal

0165-3806 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00214-0

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distribution in small and large granules [1]. A close temporal relationship between pro-TRH mRNA and TRH levels exists during development; however, whether this coincidence stems from a common developmental program is unknown. One of the experimental paradigms used for addressing the establishment of the peptide phenotype is the primary culture of fetal dissociated cells. In serum-supplemented media, fetal rodent hypothalamic cells grow in vitro for weeks; cultures develop a basal carpet of glial and ependymal cells overlaid by neuron-like cells which establish fully differentiated synapses around 10 days in vitro (DIV) [3]. After 1 week, growing neurons exhibit marked signs of advanced differentiation and secretory activity [39]. Cells display action potentials and spontaneous electrical activity [22]. Fetal rodent hypothalamic cells can also be cultured in an entirely chemically defined medium; a pattern of neuronal development similar to that obtained in serum-supplemented culture is observed but the basal carpet of non neuronal cells and the neurite network are reduced [14]. Studies in serum-free media established the importance of various factors for the differentiation of hypothalamic pro-TRH neurons [11,18,24,30]. However, either in serumsupplemented or serum-free media, TRH levels per mouse or rat hypothalamic equivalent range from 1 to 6 pg during the first week, to 20–50 pg during the third week [12,13,17,24,36]. These values are low compared to in vivo studies at equivalent time points [16,20,28]. The ratio of TRH / pro-TRH levels increases progressively during in vitro development but it is lower than that observed in vivo [17]. The discrepancy between in vitro and in vivo data could be due in part to the lack of unidentified differentiating agents in vitro leading to a deficient biosynthesis, processing or storage. We have previously demonstrated, in serum-supplemented culture, that homologous conditioned medium (CM) enhances the survival of hypothalamic neurons as well as their levels of TRH [7]. Supply of the PAM cofactor, ascorbic acid, enhances TRH levels in vitro by promoting conversion of TRH-gly to TRH [11,16,34]. Addition of bromodeoxyuridine to primary cultures of fetal diencephalic neurons increases intracellular TRH and proTRH mRNA levels; this effect was ascribed to a differentiating action on neurons [4]. Despite incorporating some of these modifications, TRH levels in vitro are still relatively low [34]. The goal of this study was to determine if the low and steady levels of TRH detected during the first week in culture are due to a delayed pro-TRH gene expression. Pro-TRH mRNA and TRH levels were determined through culture development in serum-supplemented cultures of dissociated hypothalamic cells from 17-day-old embryos. Development of pro-TRH mRNA levels was similar to that observed in vivo, and faster than that of the processed peptide, TRH.

2. Materials and methods

2.1. Primary neuronal cell cultures Hypothalamic tissue was dissected and dissociated from Wistar rats’ embryos at 17th day of gestation as previously described [41]. Cultures conditions were followed as reported [33]. Pregnant females were anesthetized with pentobarbital (33 mg / kg b.w.). The embryos were removed one by one, the brain excised, the hypothalamus dissected under a stereoscopic microscope (limits for hypothalamic dissection: posterior to the optic chiasma, anterior to the mammillary bodies, along both lateral sulcus and 3 mm in depth excluding the thalamus) and placed in Hank’s solution. Hypothalami (3 / ml) were incubated for 15 min with trypsin (0.25 mg / ml) and DNAse (0.28 mg / ml) and then, mechanically dispersed by ten passages through a Pasteur pipette in supplemented Dulbecco’s modified Eagle’s medium (S-DMEM). Supplements: 10% fetal bovine serum (Gibco-BRL), 0.25% glucose (Sigma), 2 mM glutamine (Sigma), 3.3 mg / ml insulin (Sigma) and antibiotics-antimycotic (50 units / ml penicillin G sodium, 50 mg / ml streptomycin sulfate and 0.125 mg / ml amphotericin B as fungizone; Gibco-BRL). The cell suspension was centrifuged at room temperature for 4 min at 450 g and the pellet resuspended in S-DMEM. Average yield was 2.760.5310 6 cells / hypothalamus; viability, monitored by trypan blue exclusion, was 9163% (mean6S.E.M., n540). Cell culture dishes (Costar) were pre-coated with poly-D-lysine (molecular weight 30,000– 70,000; Sigma). 2.7310 6 cells were plated on 35 mm dishes in a final volume of 2 ml of S-DMEM. On the fourth day of culture, cytosine arabinoside (10 25 M; Sigma) was added to inhibit cell proliferation. Starting at 6 DIV and every second day, half of the incubation medium was replaced with 1 ml of fresh S-DMEM. Cultures were maintained in a REVCO incubator at 378C in humidified air / 7% CO 2 for up to 18 DIV.

2.2. Hypothalamic sampling Wistar rats were used in all experiments. Rats kept under controlled lighting conditions (lights on from 7:00 to 19:00) and fed ad libitum were killed as above or by decapitation (for postnatal animals) around 12:00. Fetal or post-natal hypothalami were excised and immediately frozen on dry ice. The Society for Neuroscience (USA) ‘Guidelines for the use of animals in neuroscience research’ were followed.

2.3. RNA extraction Total RNA was extracted from cultured cells or from hypothalamic tissue by the acid guanidinium isotiocyanate method as described previously [8]. The final RNA concentration was determined by absorbance at 260 nm in

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a DU 650 spectrophotometer (Beckman). Samples that gave a 260 / 280 nm absorbance ratio .1.8 were used as template for the RT-PCR reactions.

2.4. Optimization of semi-quantitative RT-PCR for proTRH mRNA We determined the conditions to semi-quantify pro-TRH mRNA levels in primary hypothalamic cell cultures. RNA from 18 DIV cultures was used for optimization. To correct for variations in reaction efficiencies during RT-PCR reactions, the pro-TRH cDNA was coamplified with the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA, a ‘housekeeping’ gene chosen as an internal control against which pro-TRH-PCR products were normalized. Increasing amounts of total RNA (from 0.5 to 4 mg) were initially tested. Since 1 mg allowed detection of pro-TRHPCR product in an exponential range of the amplification curve (data not shown), this amount was used in subsequent experiments. Sequential series of PCR reactions were performed initially to determine the number of cycles (20–40) required to visualize pro-TRH- and G3PDH-PCR products on ethidium bromide (EthBr)-stained agarose gels and, to remain within the exponential phase of the amplification curve. Under our reaction conditions, 30 cycles were found to be appropriate for pro-TRH and G3PDH cDNAs (Fig. 1A,B). Duplex amplification reactions were carried out using the pro-TRH specific primers simultaneously with different concentrations (10–100 ng / ml) of G3PDH specific primers. As shown in Fig. 1C, 100 ng / ml and 15 ng / ml of primer concentrations were adequate to amplify both pro-TRH and G3PDH cDNAs, respectively. Size of the products was as expected; if reverse transcriptase was omitted during cDNA synthesis, there was no PCR product, ruling out the possibility that PCR products came from genomic DNA contamination (not shown).

2.5. RT-PCR measurements 1 mg of total RNA was transcribed in a final volume of 30 ml containing: 1X first strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 ), 10 mM DTT, 200 mM deoxynucleotide triphosphates (dNTPs), 100 units of M-MLV reverse transcriptase (Gibco-BRL) and 500 ng oligo (dT) 15 (Boehringer). Total RNA was pre-incubated 5 min at 658C prior to cDNA synthesis. The reverse transcription reactions were carried out for 2 h at 378C. Samples were cooled at 48C for immediate PCR or stored at 2208C until use. PCRs were performed in 50 ml reaction volumes containing: 6 ml of RT reaction product, PCR buffer (10 mM Tris–HCl, 1.5 mM MgCl 2 , 50 mM KCl, pH 8.3), 200 mM of each dNTP (in addition to the dNTP left over from the RT reaction), 2.5 units of Taq DNA polymerase (Boehringer), 100 ng / ml and 15 ng / ml of specific primers

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for pro-TRH and G3PDH cDNAs respectively. A 670 bp fragment corresponding to nucleotides 109–779 of rat pro-TRH cDNA [42] was amplified with pro-TRH primers [sense: 59-GGACCTTGGTTGCTGTCGACTCTGGCTTTG-39; antisense: 59-ATGACTCCTGCTCAGGGTCATCTAGAAGGGCT-39]. G3PDH primers [sense: 59-TGAAGGTCGGTGTCAACGGATTTGGC-39; 59CATGTAGGCCATGAGGTCCACCAC-39 (antisense)] allowed amplification of a 983 bp fragment corresponding to nucleotides 35–1017 of rat G3PDH cDNA [40]. Amplification was performed in a Robocycler (Stratagene) for 30 cycles. Each PCR cycle consisted of a heat-denaturing step at 948C for 1 min, a primer-annealing step at 608C for 1 min, a polymerization step at 728C for 1 min, and a final extension for 15 min at 728C. Aliquots of PCR reaction products (10 ml) were electrophoresed through 2% agarose gels (Ultra-pure, Bio-Rad) containing 0.2 mg / ml of EthBr. Gels were illuminated with UV light, photographed using TRI-X pan film (Kodak), and analyzed by computerized densitometric scanning of the images using a soft laser scanner Biomed and the Zeineh program. The intensities of the EthBr fluorescence signals were determined from the area under the curve of each peak.

2.6. Northern blot analyses Northern blots were performed and analyzed as previously described [41].

2.7. Radioimmunoassay of TRH After incubation, cells were rinsed with PBS and kept frozen; hypothalamic cells were scrapped with 1 ml of 1 N acetic acid / 50% methanol for TRH extraction; the suspension was kept overnight at 2208C in a tube, then centrifuged at 2000 g, and the supernatant evaporated. A previously characterized antibody (R2) [19] was used for radioimmunoassay as described [7]. Sensitivity of the assay (90% B / B0) was 10 pg.

2.8. Protein determination Proteins present in the acid–methanol precipitate were hydrolyzed overnight with 500 ml of 1 N NaOH [7] and quantified according to Lowry et al. [26].

2.9. Statistical analysis Data represent the mean6S.E.M. values from at least two different cultures. Statistical analysis was performed by ANOVA followed by least-significant-difference multiple comparison test. Significance was determined at P, 0.05.

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Fig. 1. Determination of optimal PCR cycle number and primer concentrations for duplex amplification reactions for pro-TRH and G3PDH cDNAs. All reactions were performed using the same RT and PCR conditions except for cycle numbers and G3PDH primer concentrations. One mg of total RNA from 18 DIV hypothalamic cells was reverse transcribed for 2 h at 378C, followed by 20 to 40 cycles of PCR at 948C for 1 min, at 608C for 1 min and at 728C for 1 min. (A) PCR amplifications with specific oligonucleotide primer pairs for pro-TRH (100 ng / ml). (B) PCR amplifications with specific oligonucleotide primer pairs for G3PDH (20 ng / ml). (C) Duplex PCR amplifications with different G3PDH primer concentrations (10–100 ng / ml) and a constant pro-TRH primer concentration (100 ng / ml). In each panel, left side depicts representative photographs of the ethidium bromide-stained gels of PCR products. The right side is the result of scanning stained DNA using computer-assisted densitometry and plotting the data (pro-TRH cDNA signal, panel A; G3PDH cDNA signal, panel B; or pro-TRH cDNA over G3PDH cDNA signals, panel C); each value is the mean6S.E.M. (n55, 3 independent cultures).

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3. Results

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about one fifth of cells as revealed by phase contrast microscopy analysis after 18 DIV (not shown).

3.1. Morphological development in vitro 3.2. Biochemical development in vitro Analysis by phase contrast microscopy showed that cells underwent morphological differentiation; they attached in clusters and extended neurites during the first 2 days. A mixed cell population developed. At 4 DIV a monolayer of flat cells spread over the dish forming a continuous background layer over which grew small refringent cells with an ovoid perikaryon. By 10 DIV, presumptive neurons sent out an extensive network of processes. Cultures were confluent by 18 DIV and neuron-like cells developed large phase bright cell bodies. The neuron-like population was

To determine the developmental pattern of pro-TRH expression in culture, total RNA, proteins, TRH, G3PDH and pro-TRH mRNAs levels, were quantified at each DIV. Yield of RNA per dish varied from 861 mg (n54) at 1 DIV to 5162 mg at 18 DIV (n54) (Fig. 2A). G3PDH mRNA levels per mg RNA were not altered during in vitro development, compared to 1 DIV (1 DIV: 10066%; 9665% at 18 DIV; Fig. 2B). As shown in Fig. 2B, the ratio of pro-TRH over G3PDH mRNA levels varied during

Fig. 2. In vitro developmental changes in the levels of proteins, RNA, TRH, pro-TRH and G3PDH mRNAs. (A) Protein (m) and total RNA (s) values were determined as described in Materials and methods. (B) Pro-TRH and G3PDH mRNA levels were determined by RT-PCR from a constant input of total RNA. Photographs of the ethidium bromide-stained gels of PCR products were scanned using computer-assisted densitometry. G3PDH mRNA signals X total RNA per dish (d), and pro-TRH cDNA over G3PDH cDNA signals (s), are plotted. The upper part of the panel depicts a representative photograph of the ethidium bromide-stained gel of PCR products. Each lane corresponds to a distinct DIV, from DIV 1 (left) to DIV 18 (right), except for DIV 15. (C) TRH values (♦) were determined by RIA; total pro-TRH mRNA values (h) are total RNA values X pro-TRH over G3PDH cDNAs ratio X G3PDH cDNA per mg RNA. Each value is the mean6S.E.M. (n53–4, 3 independent experiments). Statistical analysis was performed by ANOVA followed by least significant difference multiple comparison test: *P,0.05, **P,0.01, ***P,0.001 against ratio of pro-TRH cDNA over G3PDH cDNA signals at DIV 1.

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the culture time; a small increase was observed between 3 and 9 DIV, when compared with 1 DIV. A transient decrement in pro-TRH expression was observed at 10–11 DIV. A second plateau was observed between 12 and 18 DIV with the highest ratio at 13 DIV (2.2 fold vs. 1 DIV). Between 7 and 14 DIV, a 50% increase was detected. This pattern was consistent with the analysis of independent cultures by Northern blot: pro-TRH mRNA / mg RNA increased from 100% at 7 DIV to 212132% at 14 DIV (n54 independent experiments). Between 3 and 6 DIV, a second RT-PCR band of higher molecular weight was reproducibly amplified (Fig. 2B, upper panel); its size (1100 bp) corresponded to a pro-TRH mRNA containing unspliced intron number 1 [21] and was not a product of genomic DNA amplification. To calculate the relative variations of pro-TRH mRNA levels per dish during development, we multiplied RNA values per dish by the ratio of pro-TRH over G3PDH cDNAs, since G3PDH values per mg total RNA were constant. Pro-TRH mRNA levels per dish increased (without a lag period but with a transient drop at 11 DIV) 9 fold between 1 and 13 DIV, when a plateau was reached (Fig. 2C). The increase of RNA level per dish was concomitant with the increase in protein levels (Fig. 2A). In contrast, TRH levels were relatively constant from the beginning of the culture up to 8 DIV, increasing steadily afterwards until values peaked at 16 DIV (Fig. 2C).

3.3. Development in vivo To compare the development of pro-TRH mRNA levels observed in vitro with the in vivo pattern, pro-TRH mRNA levels were measured during in vivo development by semi-quantitative RT-PCR. Yield of RNA varied from 1562 mg per hypothalamus at gestational day 17, to 6962 mg at postnatal day 17 (Fig. 3A). G3PDH mRNA levels per mg RNA did not vary during in vivo development, between E17 (100625%) and P17 (10162%) (Fig. 3B). Pro-TRH mRNA was detected since E17; the ratio of pro-TRH / G3PDH cDNAs decreased during the last 2 days of gestation and closely after birth increasing at P4 to values similar, or higher, than those at E17 (Fig. 3B). Pro-TRH mRNA levels per hypothalamus increased 4 fold, from E17 to P14 (Fig. 3C).

4. Discussion Analyses of the developmental expression of TRH biosynthesis in vitro have shown that TRH levels per dish, in serum-free or -supplemented media, increase poorly with time in culture, after a lag period of a few days [13,24,36]. The goal of this study was to compare the developmental pattern of TRH and pro-TRH mRNA levels

in primary cultures of fetal dissociated hypothalamic cells to study whether the delayed increase in TRH levels found in vitro is due to a deficiency in the early steps of TRH biosynthesis. The time of tissue dissection corresponds to a time when various hypothalamic nuclei express pro-TRH mRNA. In culture, pro-TRH mRNA per dish accumulated at a rate similar or higher than observed in vivo. The initial increase in pro-TRH mRNA expression may be due, at least in part, to enhanced transcription as part of the intrinsic program of differentiation of the neurons and / or to paracrine influences. Enhanced transcription was compatible with the observed transient accumulation of a putative intronic pro-TRH mRNA. In contrast, in vitro TRH levels, stable during the first week and raising during the 2nd week, were still very low (110–150 pg per hypothalamic equivalent at 16–18 DIV) compared to in vivo values at an equivalent time point (3000–4000 pg / hypothalamus, at P16 [28]). This differs from in vivo evidence that TRH [10,16,20,28] and proTRH mRNA [9, this study] levels per hypothalamus increase before and after birth. Therefore, post-transcriptional events such as translation, processing, or storage, seem to require additional time and as yet unidentified factors for complete maturation in vitro. Various possibilities related to external clues may account for the in vitro delayed increase in TRH levels. For example, a delayed synaptic input since synapse establishment requires more than 1 week in culture [3,22]; or, inadequate auto-, para- or endocrine influences or cell–cell or extracellular matrix (ECM) clues. Previous studies in our laboratory have demonstrated that homologous CM enhances expression of TRH in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. The CM component(s) regulating TRH expression is probably of glial origin [7]. In serum-free cultures, a soluble factor such as brain neurotrophic factor, but not neurotrophin-3, enhances pro-TRH mRNA levels [18]; while, coating dishes with ECM proteins increases proTRH but not pro-TRH mRNA levels [31]. In regard to the specific steps for TRH biosynthesis that may be deficient in vitro, reduced translation of pro-TRH mRNA cannot be discarded [31]. However, Grouselle et al. [17] have shown that the ratio of TRH / pro-TRH immunoreactivity increases between the first week and subsequent weeks in vitro and suggested this was due to a later acceleration of pro-TRH processing. Post-translational modifications among peptides include endoproteolytic cleavage by protein convertases. PCl and PC2 are two convertases present in pro-TRH hypothalamic neurons [35,38] that process pro-TRH in vitro [29]. Their mRNAs are detected in hypothalamus since E13 [43]. Increasing amounts of active PC1 are observed later in development (O. Vindrola, personal communication). PC1 mRNA is present in part [34] or all [30] of fetal hypothalamic pro-TRH neurons in culture. It remains to be established whether particular requirements for full expression of the

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Fig. 3. In vivo developmental changes in the levels of RNA, pro-TRH and G3PDH mRNAs. (A) RNA (s) values per hypothalamus were determined as described in Materials and methods. (B) Pro-TRH and G3PDH mRNA levels were determined by RT-PCR from a constant input of total RNA. Photographs of the ethidium bromide-stained gels of PCR products were scanned using computer-assisted densitometry. G3PDH mRNA signals X total RNA per dish (♦) and pro-TRH cDNA over G3PDH cDNA signals (s) are plotted. The upper part of the panel depicts a representative photograph of the ethidium bromide-stained gel of PCR products. Each lane (from left to right) corresponds to the stage indicated in the lower part of the graph; F17–21, P 3, 4, 6, 7, 11, 13, 14 and 17. (C) Total pro-TRH mRNA values (s) are total RNA values X pro-TRH over G3PDH cDNAs ratio X G3PDH cDNA per mg RNA. Each value is the mean6S.E.M. (n53–4, 3 independent experiments). Statistical analysis was performed by ANOVA followed by least significant difference multiple comparison test: *P,0.05, **P,0.01, ***P,0.001 against ratio of pro-TRH cDNA over G3PDH cDNA signals at F17.

convertases and their activity are still lacking in culture conditions (see for example [31]). The final processing of TRH precursor involves the conversion of TRH-gly to TRH by PAM, an enzyme activity whose developmental expression is similar in vitro and in vivo [11], and requires molecular oxygen, ascorbate, Cu 21 and Zn 21 , for optimal activity. Addition of ascorbate increases TRH levels in cultures of fetal rat hypothalamic cells, either serum-free or serum-supplemented [11,16]. Under our culture conditions ascorbic acid treatment increased TRH content, but only 2 fold [34] in spite of the use of a maximally effective ascorbate concentration during the complete culture period. It is therefore unlikely that failure to accumulate TRH is only due to an inadequate PAM activity.

Insufficient accumulation of TRH may also stem from failure to retain the processed peptide due to an immature secretory system. The ability to secrete TRH in response to a depolarization with high KCl concentration occurs only during the second week, either in serum-free or serumsupplemented cultures [23,25]. The spontaneous release of TRH in the culture medium is high compared to the cell content, either in serum-free or serum-supplemented cultures [23,24,27]. A high in vitro release from cell cultures, compared for example to tissue slices from adult animals [19], does not seem exclusive of TRH neurons since other cultured fetal peptidergic neurons show this behavior [2,15,37]. It remains to be studied whether important clues are still lacking for an adequate differentiation of the secretory apparatus.

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In conclusion, pro-TRH mRNA levels in culture follow a pattern similar to the in vivo situation. We suggest that the initially low and steady levels of TRH detected in primary cultures of fetal hypothalamic cells are not due to a deficient transcription of pro-TRH gene since pro-TRH mRNA levels follow a similar pattern as observed in vivo. Neurons in culture might lack adequate maturity in some steps of the post-transcriptional events or in their secretory apparatus. During development of peptidergic systems, regulation of peptide mRNA levels may occur independently of the down-stream steps leading to peptide accumulation. This system might provide a good model for defining the factors involved in the sequential establishment of peptide phenotype in post-mitotic neurons.

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Acknowledgements The authors express their gratitude to M. Cisneros, F. Romero and M. Villa for technical support as well as to E. Mata and S. Gonzalez for providing the animals used in this study. Supported in part by grants from DGAPAUNAM IN216496, CONACYT-25386-N and 33351 and EU CI1*-CT93-0301.

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