Tyrosine Hydroxylase Expression in Differentiating Neurons of the Rat Arcuate Nucleus: Stimulatory Influence of Serotonin Afferents

The influence of serotonin afferents on tyrosine hydroxylase expression in differentiating neurons of the rat arcuate nucleus was studied in vivo and in vitro. In the in vivo study, pchlorophenylalanine inhibited serotonin synthesis in fetal brain from the 11th to the 20th embryonic day. We then used semiquantitative immunocytochemistry to evaluate tyrosine hydroxylase levels in neurons of the arcuate nucleus in fetuses at the 21st embryonic day or in offspring at the 35th postnatal day. Serotonin depltion significantly decreased the tyrosine hydroxylase content in neurons of males and females at the 21st embryonic day and in males at the 35st postnatal day. For the in vitro study, embryonic neurons of the arcuate nucleus were cocultured with embryonic neurons of the raphe nucleus, the main source of serotonin innervation of the brain, including the arcuate nucleus. Co-culture of the neurons resulted in a genderspecific increase of the tyrosine hydroxylase level in the neurons of the arcuate nucleus. In turn, the neurons of the raphe nucleus showed increased levels .of serotonin in both males and females, with no sexual dimorphism. Thus, our results suggest a stimulatory, long-lasting effect of serotonin afferents on tyrosine hydroxylase expression in the differentiating neurons of the rat arcuate nucleus during prenatal ontogenesis.


INTRODUCTION
Since the initial detection of dopamine in neurons of the arcuate nucleus and their axon terminals in the median eminence with the histofluorescence technique (Dahlstr6m & Fuxe, 1964;Fuxe, 1964;Bj6rklund & Nobin, 1973), the socalled tuberoinfundibular dopaminergic system has attracted the particular attention of neurobiologists and neuroendocrinologists. This interest is explained by the great contribution of the tuberoinfundibular dopaminergic system to the inhibitory control of the adenohypophysial prolactin secretion and, thereby, to the regulation of reproduction (Neill, 1988). In turn, in adults, the dopaminergic neurons of the arcuate nucleus are under complex regulation by intercellular signals like prolactin, estrogens, testosterone, serotonin, enkephalin, galanin, neuropeptide Y (Moore et al., 1985;Kiss & Halisz, 1986;Arbogast & Voogt, 1991Hrabovszky & Liposits, 1994;Magoul et al., 1994;Hong et al., 1995). Arbogast and Voogt suggested that the same intercellular messengers might influence the differentiation of dopaminergic neurons of the arcuate nucleus during ontogenesis. Although the genesis and differentiation of dopaminergic neurons in the arcuate nucleus have been studied in many species (Halpem-Sebold et al., 1985;Daikoku et al., 1986;Friedman et al., 1989;Ugrumov et al., 1989;Richards et al., 1990;Romero & Phelps, 1993;Phelps et al., 1994;Balan et al., 1996Balan et al., , 2000, few data are available on their regulation by intercellular signals (Friedman et al., 1989;Romero & Phelps, 1993;Phelps et al., 1994). The aim of the present study was to evaluate the possible influence of serotonin afferents on differentiating dopaminergic neurons of the arcuate nucleus; namely, on the expression of tyrosine hydroxylase (TH), the first, rate-limiting enzyme of dopamine synthesis. Serotonin afferents appear to be among the potential regulators of the differentiation of dopaminergic neurons as 1. dopaminergic neurons in adults display serotonin receptors and are synaptically innervated by serotonin fibers belonging to the neurons of the raphe nucleus (Fuller & Clemens, 1981;Steinbusch & Nieuwenhuys, 1981; Kiss & Halfisz, 1986;Willoughby et al., 1988;Jorgensen et al., 1993); 2. serotonin is generally considered a putative morphogen that is functionally active before synaptogenesis and the onset of neurotransmission (Lauder et al., 1980;Lauder, 1983Lauder, , 1990Ugrumov et al., 1994;Ugrumov, 1997); and there is at least a timing correlation between the early differentiation of dopaminergic neurons (Daikoku et al., 1986;Ugrumov et al., 1989;Balan et al., 1996Balan et al., , 2000 and the initial arrival of serotonin fibers in the arcuate nucleus (Lidov & Molliver, 1982;Aitken & Tork, 1988).

Pharmacological model of serotonin depletion
Wistar pregnant rats were injected intraperitoneally daily with pCPA in saline (100 mg/kg b. wt.), an inhibitor of serotonin synthesis, from d 11 to d 20 of pregnancy (day of conception being the first embryonic day; El). Untreated control animals received saline over the same period. Pregnant rats on d 21 of gestation or their offspring on postnatal d 35 (the day of parturition being the first postnatal day; P1) were anesthetized with pentobarbital (50 mg/kg b. wt.); the fetuses were removed for subsequent processing. On E21, a minimum of four treated males and four untreated control males and the same number of females from two pregnant rats were processed for immunocytochemistry. The same number of pCPA-treated and control males and females at P35 were used from a minimum of two litters.

Fixation and immunocytochemistry
All reagents were from Sigma unless otherwise noted. The fetuses and young rats were perfused through the heart, first with saline for 2 to 5 min at 37C and then fixed for 15 min with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4C. The brain was then postfixed for 2 h by immersion in paraformaldehyde at 4 C, rinsed for h in 0.02 M phosphate buffer and 0.9% NaCI (PBS), pH 7.2-7.4, at 4 C, immersed overnight in 15% sucrose PBS at 4 C; and then frozen in isopentane at-35 C. Serial coronal sections of the mediobasal hypothalamus (arcuate nucleus and median eminence) were cut into 12-m-thick sections with a cryostat microtome at-20 C, thaw-mounted on slides covered with gelatine, and quickly dried on a slide warmer. Sections of the brains from pCPA-treated and control males and females of the same age were mounted on the same slide and processed together.
The specificity of the antiserum raised against TH was controlled for by Arluison et al. (1984) and in the present study by omitting the primary antibody. After each incubation (except the second), the sections were rinsed with PBS. All incubations were carried out in a humid chamber. The sections were rinsed with 0.05 M Tris-HCl buffer, pH 7.6; the peroxidase reaction of the avidin-biotin complex was developed in Tris-HCI buffer containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide. The developing time was constant for each age studied. Finally, the sections were dehydrated and coverslipped. Dissociated primary culture At the 17 th day of gestation, the pregnant rats were decapitated, and the fetuses were removed aseptically. Gender was determined by revision of the gonads, and male and female materials were processed separately. The fetuses were decapitated, and the brain was removed from the skull. The central rhombencephalon with the raphe nucleus was dissected using mesencephalic and cervical flexures as landmarks (Konig et al., 1987). The wedge of tissue was removed just caudal to the mesencephalic flexure (rostral to the rhombencephalic isthmus). For dissection of the mediobasal hypothalamus, the brain was placed on its dorsal surface, and frontal cuts were made just caudal to the pituitary stalk and rostral to the primary portal plexus. Finally, the hypothalamic area around the bottom of the third ventricle was dissected from the thick frontal slice, and the meninges were removed (Melnikova et al., 1999).
The dissected tissues, were used to prepare dissociated primary cultures as described before (Melnikova et al., 1999;Tixier-Vidal & Faivre-Bauman, 1990). For this purpose, mediobasal hypothalamus and raphe nucleus from males and females were pooled separately and dissociated mechanically in F12/DMEM medium (Gibco), supplemented with 10% fetal calf serum. The cell suspension was centrifuged for 10 min at 800 rpm and then resuspended in serum-free F12/DMEM containing transferrin (100 9g/mL), putrescin (10 -4 M), insulin (5 tg/mL), glutamine (5 x 10 -3 M), sodium selenite (2 x 10 -5 M), progesterone (2xl 0 -s M), 17-13-estradiol (10 M), corticosterone (10 -7 M), triiodothyronine (10 .8 M), HEPES (3.57 g/L), sodium bicarbonate (2.44 g/L) plus arachidonic acid (1 9g/mL), and docosahexaenoic acid (0.5 9g/mL), adsorbed on bovine serum albumin (fatty acid free, 37.5 Ixg/ mL). The cell suspension, at a density of 500,000 cells/cm:, was placed onto poly-L-lysine-coated 22-mm plastic coverslips in 60-mm diameter Petri dishes (2 coverslips per dish). Cell suspensions from the mediobasal hypothalamus and the raphe nucleus were plated on separate coverslips. The coverslips with mediobasal hypothalamus and raphe nucleus were placed into the same Petri dish for co-culture and into different Petri dishes for single cultures. The cell survival in the primary cell suspension usually exceeded 85%, estimated by trypan blue exclusion in a hemocytometer. All procedures described above were made in sterile conditions. The cultures were maintained for 7 d at 37C in a humidified atmosphere of 5% CO. The incubation medium supplemented with cytosine arabinoside (10 -s M), an inhibitor of glial cell proliferation, was renewed on the 3 and 5 th days. On the 7 th day, the cultures were fixed for 15 min with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at room temperature and then rinsed with 0.02 M PBS. Cultures of mediobasal hypothalamus and raphe nucleus were then immunostained for TH or for serotonin, respectively, using the avidin-biotin technique, as described for the cryostat sections, but with 4% normal goat serum replacing the 1% serum. Antiserum to serotonin was diluted 1:2000.
The specificity of the antiserum was controlled for by Tramu et al. (1983) and in this study by omitting the primary antibody.

Semi-quantitative image analysis
Carto, (R) a software program developed by IMSTAR (France), was used for the semiquantitative analysis of TH-immunoreactive material in the cell bodies of the arcuate nucleus at E21 and P35, as well as of serotonin and TH-immunoreactive materials in culture and co-culture of the mediobasal hypothalamus and raphe nucleus. Using this approach, we could accurately assess relative amounts of substrates and easily compare the results of different experiments. Indeed, all preparative procedures of the sections and cultures were performed under standardized conditions, allowing meaningful quantitative analysis (Smolen, 1990).
Each section or culture was reviewed under a light microscope with a x25 objective, followed by the transfer of its images through a video camera (CCD) to a computer monitor (Pentium 60; IBM, Armonk, NY, USA) and subsequent recording.
The profiles of the individual immunoreactive cell bodies were outlined with a light pen to get a morphometric mask, and the relative amounts of immunoreactive materials were measured as 'gray level' (GL) and related to the optical density (OD) of the specimen as follows (Smolen, 1990): The GL of the background was measured in the vicinity of the mediobasal hypothalamus with no specific immunostaining on sections or in the immunonegative areas in culture.
[3HlSerotonin uptake The hypothalamus with the adjacent septum and diagonal band, corresponding to neuroendocrine centers with an important serotonin innervation (Steinbusch & Nieuwenhuys, 198 I;Montange & Calas, 1988), was dissected in 21-d-old fetuses treated with pCPA or saline, as described above. As gender differences have not been found in the serotonin system of rat fetuses (Lidov & Molliver, 1982;Wallace & Lauder, 1983), we evaluated male and female nervous tissues together. Tissue fragments were incubated in Krebs-Ringer bicarbonate medium (pH 7.4) composed of (raM): NaCI (120), KCI (4.8), CaCI2 (2), MgSO (1.2), KH_,PO (1.25), NaHCO3 (25), D-glucose (10). The solution also contained 0.3 mM pargyline, 0.13 mM ascorbic acid, and 2.4 mM EDTA. The tissues were incubated twice for 10 min each at 37 C in medium saturated with a mixture of 95% oxygen/5% carbon dioxide. The third incubation was carried out in medium containing 25 x 10 -9 M [3H]serotonin (17)(18)(19)(20) Ci/mM, Amersham, England). We stopped the [3H]serotonin uptake by cooling the medium to 0 C. The materials were rinsed several times in pure, ice-cold medium, gently dried, weighed, and transferred to vials containing mL 96% ethanol. The vials were then incubated for 18 h to extract the radioactively labeled compounds. Thereafter, the solution was dissolved in 10 mL of liquid scintillator and radioactivityexpressed as the number of scintillations/min (cpm) per mg nervous tissue was measured in a liquid scintillation counter (Intertechnique SL-30, France). Unspecific binding was evaluated by pre-incubating samples in medium containing an inhibitor of serotonin uptake, citalopram (10 -5 M, Landbeck, Denmark), before the incubation in [3H]serotonin-containing medium.
Specific uptake of the label was estimated as the difference between [3H]serotonin binding with and without pre-incubation with citalopram.

Statistics
Statistical analysis was carried out using descriptive statistics, the nonparametric Wilcoxon's test, and the Student's t-test for unpaired data.

In vivo study
Fetuses: at E21, most TH-immunoreactive neurons were located in the ventrolateral region of the arcuate nucleus, although a few were also found in the dorsomedial region (Fig. 1). The THimmunoreactive neurons, mainly unipolar or bipolar, were small, pCPA treatment of pregnant rats caused a decrease in the OD of TH-immunoreactive neurons of the arcuate nucleus in male and female fetuses, with no gender difference (Fig. 2).
Young rats: at P35, the number of TH-immunoreactive neurons appeared to increase and were distributed predominantly in the dorsomedial region of the arcuate nucleus and, to a lesser degree, in the ventrolateral region. Decreased levels of THimmunoreactive material was found in neurons of the arcuate nucleus only in the male offspring of pCPA-treated pregnant rats (Fig. 3). Although the same tendency was characteristic of females, no significant difference was observed.

In vitro study
The appearance of TH-immunoreactive neurons of the mediobasal hypothalamus in culture was similar to that in vivo. Such neurons were small, unipolar or bipolar, with a relatively large nucleus and scanty cytoplasm (Fig. 1). Rare multipolar TH-immunoreactive neurons were also observed.
Serotonin-immunoreactive neurons were larger, with unbranched dendrite-like processes and highly arborized, axon-like processes. The presumed axons were identified according to the criteria of Konig et al. (1987)" relative thinness, uniform caliber throughout the entire length, collaterals (if present), branching at angles close to 90 All other processes were considered presumptive dendrites.
In single culture of the mediobasal hypothal-amus, the OD of TH-immunoreactive neurons in females exceeded that of males (0.185 + 0.012 in females vs. 0.14 + 0.015 in males, P < 0.05). The OD of serotonin neurons in single cultures of the raphe nucleus did not show sexual dimorphism (Fig. 4). Co-culture of mediobasal hypothalamus and raphe nucleus resulted in an increase in the OD of THimmunoreactive neurons in females and to a greater extent in males (0.134 + 0.017 males vs 0.05 + 0.014 females, P < 0.05). Moreover, co-culture resulted in an increase in the OD of serotonin immunoreactive neurons in both males and females, without gender differences (Fig. 4).

DISCUSSION
The pharmacological model of serotonin depletion in the fetal brain induced by intraperitoneal injections of pCPA to pregnant mothers was used in this and in prior studies for evaluating the influence of serotonin on the genesis and differentiation of target neurons (Lauder et al., 1981;Lauder, 1983;Ugrumov et al., 1994;Ugrumov, 1997). The specific effect of pCPA is related to a decrease of tryptophan hydroxylase activity and serotonin content in the fetal brain (Lauder et al., 1981(Lauder et al., , 1985Lauder, 1983). In contrast to pCPA concentrations used in earlier studies, (Koe & Weissman, 1966;Lepetit et al., 1991;Tagliamonte et al., 1973), the at least 2.8fold lower concentration (100 mg/kg b. wt.) used here was sufficient to provoke serotonin depletion, with a minimal risk of side effects (Lauder et al., 1981;Lauder, 1983). Finally, we used the pharmacological model of serotonin depletion to evaluate the immediate and delayed effects on TH expression in differentiating neurons of the arcuate nucleus. In addition to the in vivo pharmacological model, we used in vitro co-cultures of the mediobasal hypothalamus and the raphe nucleus to determine whether serotonin afferents modify TH expression in differentiating neurons of the arcuate nucleus. The separation of arcuate nucleus and raphe nucleus neurons during the whole period of culture facilitated their identification and made possible the study of mutual regulatory effects under the absence of cell-cell contact.
To compare TH expression in the neurons of the arcuate nucleus in both experimental and control animals, we used a semi-quantitative image analysis, measuring the OD of the immunoreactive material correlated to the TH content. We emphasize (see Experimental Methods) that for the semiquantitative analysis, we processed male and female materials under standardized conditions (fixation, cutting, and immunostaining of sections mounted on the same slides). The validity of this approach was proved in early technical studies (Smolen, 1990) and later confirmed by concrete applications (Beltramo et al., 1994(Beltramo et al., , 1997Balan et al., 1996Balan et al., , 2000. Still, one should take into account that we could compare only the relative amounts of immunoreactive materials in males and females at each age studied, in vivo and in culture, separately. In addition to the experimental approaches mentioned above, we used a biochemical isotopic technique to evaluate serotonin uptake by the developing hypothalamus and the adjacent neuroendocrine centers in chronic serotonin-depleted rats and in untreated control rats. This approach served to clarify whether the influence of serotonin in the arcuate nucleus of fetuses is limited to the target neurons, including TH-expressing neurons, or whether autoregulation of the local serotonin innervation also occurs. Indeed, serotonin uptake in the brain regions outside the raphe nucleus is considered an index of the density of serotonin fibers (Snyder et al., 1970;Sakurai et al., 1990).
Serotonin influence on the tyrosine hydroxylase expression in vivo According to our semi-quantitative immunocytochemical study, chronic serotonin depletion occurring over the second half of the intrauterine development significantly decreased the TH content in differentiating neurons of the rat fetal arcuate nucleus at E21. In the meantime, the observation that serotonin uptake by hypothalamic tissue remained unchanged indicates that serotonin has no influence on the development of serotonin innervation. Taken together, the data suggest that serotonin stimulates TH expression in differentiating neurons of the arcuate nucleus. The morphogenetic action of serotonin on specific synthesis in differentiating target neurons appears to be cellspecific. In contrast to TH expression in the arcuate nucleus, vasoactive intestinal polypeptide gene expression in differentiating neurons of the suprachiasmatic nucleus is under serotonin inhibitory control during the same period of ontogenesis .
Although the mechanism of the influence of serotonin on differentiating target neurons remains uncertain, apparently it is not transmitted via synaptic input because the major synaptogenesis in the arcuate nucleus occurs after birth (Koritszansky, 1981). Several investigators believe that before the onset of neurotransmission, serotonin exerts its action on the differentiating target neurons as a diffusive neurohumoral factor via serotonin recaptors that are expressed and become functionally active early in embryogenesis (Whitaker-Azmitia et al., 1987;Hellendall et al., 1993;Whitaker-Azmitia et al., 1996;Wu et al., 1999). The 5-HT1 and 5-HT2 receptors appear to be the most probable candidates as they are involved in the inhibitory control of dopamine neurons of the arcuate nucleus in adults, providing the regulation of adenohypophysial prolactin secretion (Meltzer et al., 1983;Willoughby et al., 1988;Jorgensen et al., 1993). In addition to a direct action of serotonin on differentiating target neurons, an indirect action via glial cells cannot be excluded. Indeed, serotonin contributes to the regulation of secretion of glialderived neurotrophic/growth factors, which, in turn, stimulate neuron differentiation (Whitaker- Azmitia et al., 1996). We approached this problem using cell culture (see below).
The decreased levels of TH observed in pCPAtreated fetuses persisted in young male rats (P35).
The data suggest that serotonin provides a longlasting gender-specific, stimulatory influence on TH expression when acting on the differentiating neurons of the arcuate nucleus during the critical prenatal period. The molecular mechanism of the imprinting effect of serotonin has to be worked out in a future study.
Influence of serotonin afferents on hydroxylase expression in vitro tyrosine According to the present study, in dissociated neurons of the arcuate nucleus, TH expression increases when co-cultured with dissociated neurons of the raphe nucleus, the only source of serotoninergic neurons in the brain. Apparently, the stimulatory effect was provided by neurohumoral factors originating in the raphe nucleus. The results of the in vitro study demonstrate that the influence of gliaderived neuro-trophic/growth factors can be excluded because the proliferation of glial cells was inhibited. In other words, TH expression in neurons of the arcuate nucleus is regulated by humoral factors that are synthesized by neurons of the raphe nucleus. Although such factors were not identified in this study, serotonin seems to be the most probable candidate, which agrees with our in vivo study and remains to be deduced in a future study.
Moreover, the results of our in vitro study revealed that the differentiating neurons of the arcuate nucleus and those of the raphe nucleus are mutually regulated. In fact, the significant increase in the serotonin content of the raphe nucleus neurons is due to their co-culture with the dissociated neurons of the arcuate nucleus. The data presented here are consistent with prior observations that the differentiation of serotonin neurons is influenced by their targets (Azmitia et al., 1990;Whitaker-Azmitia, 1991). In adults, the brain areas with dense serotonin innervation (hippocampus, cortex, caudate, olfactory bulb) stimulate the uptake of [3H]serotonin by neurons of the raphe nucleus in co-culture (Whitaker- Azmitia, 1991 ).
Sexual dimorphism in tyrosine expression and its regulation hydroxylase According to the results of our in vitro study, the TH content in neurons of the arcuate nucleus in single culture of females significantly exceeds that of males. This was also the case for neurons in the arcuate nucleus from El8 in vivo, the first age studied, onward (Balan et al., 2000). The data agree with prior observations showing that in vivo (Balan et al., 1996) and in the initial differentiation (outgrowth of processes) in culture (Reisert et al., 1989), the origin of hypothalamic TH-expressing neurons in females precedes that in males. Data from earlier studies (Reisert & Pilgrim, 1991;Reisert et al., 1994;Ugrumov, 1994;Balan et al., 1996) suggest that sexual dimorphism in TH expression can be determined by gender-specific primary genetic programs of brain development or can be provoked by gender-specific neurohumoral factors that are different from testosterone or estradiol. Prolactin, a hormone of reproduction, might be considered a candidate for the factor contributing to the gender-specific development of TH-expressing neurons of the mediobasal hypothalamus Beyer et al., 1994).
In the in vivo study presented here, two observations can be discussed in relation to sexual dimorphism of differentiating TH-expressing neurons of the arcuate nucleus. In contrast to our results in an earlier in vivo study (Balan et al., 2000) and those of the present in vitro study in intact fetuses, we failed to find gender differences in the TH content of saline-injected control fetuses. According to prior data, the schedule of the genesis of serotonin target neurons and catecholamine metabolism in different brain regions can be modified also in fetuses by saline injections into their pregnant mothers (Moyer et al., 1978;Lauder et al., 1980). Although the mechanism of action remains uncertain, it is most probably related to an adrenal reaction to chronic stress that is provoked by regular saline injections. According to another observation in our study, pCPA-induced serotonin depletion decreases the level of TH in males. Although the same tendency was a characteristic of females, the gender difference was not significant. From the data indirectly follows that testosterone somehow intensifies the long-term effect of serotonin depletion on the differentiating TH-expressing neurons. Thus we conclude that during prenatal ontogenesis, serotonin afferents provide a longterm stimulatory influence on TH expression in neurons of the rat arcuate nucleus.