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Keywords:

  • Serotonin;
  • Glucocorticoid receptor;
  • Raphe nuclei;
  • Hippocampus;
  • Primary cell culture;
  • Rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Abstract: The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamuspituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (±)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionamide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.

The hypothalamus-pituitary-adrenal axis and brain serotonin (5-HT) systems are functionally interdependent. Via mineralocorticoid receptors and glucocorticoid receptors (GRs), adrenal steroids control the activity of the raphe-hippocampal 5-HT system involved in the regulation of the hypothalamus-pituitary-adrenal axis (Meijer and de Kloet, 1998). Conversely, 5-HT or 5-HT receptor agonists acting on different 5-HT receptor subtypes stimulate adrenocorticotrophic hormone and corticosterone secretion (Bagdy et al., 1989) and regulate hippocampal corticosteroid receptors in a complex way. GRs are found in almost all serotoninergic neurons (Harfstrand et al., 1986). Serotoninergic cells located in the median and dorsal raphe nuclei innervate the frontal brain region, including the hippocampus, where they project onto pyramidal neurons, which contain high concentrations of mineralocorticoid receptors and GRs (Reul and de Kloet, 1986; Van Eekelen et al., 1988).

In the adult rat hippocampus the regulation of mineralocorticoid receptors and GRs as well their encoding mRNAs by 5-HT appears to be dependent on the duration of the 5-HT metabolism alteration, on the presence or absence of circulating corticosterone, and on the hippocampal subregion analyzed (Siegel et al., 1983; Seckl et al., 1990; Novotney and Lowy, 1995; Yau et al., 1997).

In raphe nuclei, numerous studies have reported a GR-mediated effect of corticosterone on 5-HT receptors and/or 5-HT synthesis and release (Van Loon et al., 1981; Tejani-Butt and Labow, 1994). To our knowledge, however, no data concerning the effect of 5-HT on GRs in the raphe nuclei have been reported.

In the fetus, Meaney et al. (1985) found that dexamethasone binding sites are present in the limbic brain of the fetal rat at embryonic day (E) 16. Using in situ hybridization and immunocytochemical techniques, Cintra et al. (1993) detected a GR mRNA signal at E15 and substantial GR immunoreactivity at E17 within the raphe nuclei. The functionality of GRs during fetal life has been shown in in vitro and in vivo experiments. Treatment with corticosterone was found to decrease the number of GR binding sites in primary cultures of hippocampal neurons (Vedder et al., 1993; O’Donnell and Meaney, 1994), and Reichardt and Schutz (1996) showed that GR-dependent regulation of the hypothalamus-pituitary-adrenal axis was established during fetal development around E16.5. In the early period of development (E14) Héry et al. (1999) demonstrated the presence of functional 5-HT1A and 5-HT1B receptors within the rostral raphe nuclei.

Taking into account early functionality of 5-HT and GRs and in the light of a previous report by Mitchell et al. (1990) that showed that 5-HT can modulate the number of GR binding sites in fetal hippocampal cultures, the present study was conducted to examine the effect of 5-HT and of a 5-HT reuptake inhibitor on GR binding sites in fetal hippocampus and raphe nuclei. The following questions were addressed: (a) Can GRs in fetal raphe nuclei be controlled by 5-HT receptor activation? (b) Is this type of control the same in areas containing either 5-HT cell bodies, i.e., raphe nuclei, or 5-HT terminals, i.e., hippocampus? (c) Which 5-HT receptor subtypes are involved in these controls?

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Dissection

Pregnant Sprague-Dawley rats after 14 or 19 days of gestation were anesthetized with thiopental (Pentothal), and the fetuses were removed aseptically. The day of sperm identification was designated as day 0 of gestation. Hippocampi were dissected from the fetuses removed at 19 days of gestation. The raphe nuclei were dissected from the fetuses removed at 14 days of gestation. The raphe nuclei dissection procedure has been described in detail elsewhere (König et al., 1987). In brief, the part of the head containing the mesencephalon and the rhombencephalon was dissected. Because immunocytochemical studies using antibodies directed against 5-HT have revealed that at E15 all serotoninergic raphe cells are located in the rhombencephalon (König et al., 1988), this region was freed from the mesencephalon.

Cultures

The raphe nuclei and the hippocampi were mechanically dissociated in an F12/Dulbecco’s modified Eagle’s medium (50:50 vol/vol) medium supplemented with 10% fetal calf serum, 1 mM glutamine, 0.6% glucose, penicillin (50 U/ml), and streptomycin (50 μg/ml). After being resuspended in a serum-free medium, they were seeded at a density of 1.5 × 106 cells in 1.5 ml of medium per 35-mm-diameter plastic tissue culture dish. Tissue culture dishes were pretreated as follows: They were coated with poly-D-lysine (10 μg/ml) for 12 h, rinsed with distilled water, and incubated for 2 h with 20% fetal calf serum, which was then withdrawn to dryness. The synthetic culture medium used was that described by Bottenstein and Sato (1979) supplemented with 17β-estradiol (10-12M), triiodothyronine (10-9M), arachidonic acid (1 μg/ml), and docosahexaenoic acid (0.5 μg/ml). Depending on the culture (raphe nuclei or hippocampi), cells were exposed to 5-HT, 5-HT receptor agonists and antagonists, a 5-HT reuptake inhibitor, or a monoamine oxidase inhibitor 6 days after plating for the last 1-5 days in culture, after which cells were harvested for binding assays. In each experiment, 12-18 wells were processed for each experimental group.

GR binding assay

At the time of harvesting (10 days in vitro), the medium was removed, the cultures were rinsed twice with isotonic buffer, and the cells were scraped into TEDGM buffer (30 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 10 mM sodium molybdate), pH 7.4. Three wells were pooled for each data point, and then the samples were sonicated and centrifuged at 4°C for 60 min at 100,000 g. Aliquots (150 μl) of the resulting soluble fraction were incubated with 100-μl aliquots of the radioligand in TEDGM at 4°C for 20-22 h. For measurement of GR binding capacity, [3H]dexamethasone (47 Ci/mmol; NEN) was used in a concentration range of 0.2-6 nM for saturation experiments and at a 10 nM saturating concentration in single-point assays. GR-specific binding was obtained by subtracting the binding of [3H]dexamethasone in the presence of a selective GR competitor, RU 28362 (0.5 mM), from the total binding of [3H]dexamethasone alone. Bound tritiated ligand was separated from unbound tritiated ligand in 100-μl aliquots of incubation mixture with Sephadex LH-20 (Pharmacia Fine Chemicals) columns made from disposable pipette tips and equilibrated with TEDGM. The columns were eluted with TEDGM (500 μl) into scintillation vials filled with 8 ml of scintillation cocktail, and radioactivity was counted in a Packard β-counter with an efficiency of 55% for tritium.

Protein content was determined by the method of Bradford (1976). The results are expressed as femtomoles per milligram of protein. Maximal numbers of binding sites (Bmax) and the dissociation constant (KD) were derived from saturation experiments using the Ligand program (Munson and Rodbard, 1980).

Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

After 10 days of culture, the medium was withdrawn, and cells were washed twice with 1 ml of artificial CSF (126.5 mM NaCl, 27.5 mM NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 1.1 mM CaCl2, 0.85 mM MgCl2, 0.5 mM Na2SO4, and 5.9 mM glucose) adjusted to pH 7.3 with a mixture of O2/CO2 (95:5 vol/vol). Cells were preincubated for 15 min in 1 ml of CSF and kept at 37°C in an incubator. Pargyline (10-5M) or citalopram (10-7M) was then added into the incubating medium. Incubation was stopped 30, 60, or 120 min later, and the media were collected in tubes maintained in chilled water. The collected medium was passed through Durapore filters (pore size, 0.45 μm; Millipore) before biochemical measurements. 5-HT and 5-HIAA were assayed using HPLC coupled to an electrochemical detector. The mobile phase consisted of 0.1 M sodium acetate buffer containing 6% methanol (pH 4.5). The liquid chromatographic system was composed of a model 590 Waters pump, a Perkin-Elmer ISS 100 automatic injector, and a Merck C18 column (250 × 4 mm, packed with 4-mm spherical particles). Electrochemical detection was performed using a Waters model 464 detector. 5-HT and 5-HIAA were identified and quantified by comparing their retention times and peak areas with those of external standards.

Drugs

(±)-2,5-Dimethoxy-4-iodoamphetamine (DOI) hydrochloride, (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and ketanserin were obtained from RBI (Sigma, France). Citalopram hydrobromide, WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionamide] hydrochloride, and RU 28362 were generous gifts from Lundbeck (Denmark), Wyeth Research (U.S.A.), and Roussel Uclaf (France), respectively.

Statistical analysis

The statistical significance of any difference between groups was analyzed using a one-way ANOVA followed by Scheffé’s test. Differences were considered statistically significant when p values were <0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Effects of 5-HT, 5-HT agonists, and citalopram on GR binding sites in hippocampal cell cultures

GR binding was measured at a 10 nM saturating concentration of [3H]dexamethasone using single-point assays. Under control conditions, the levels of GR binding sites were 125.3 ± 13 fmol/mg of protein.

Hippocampal cell cultures were exposed to 5-HT or to 5-HT agonists from day 6 after seeding and for the last 4 days in culture. Exposure to 5-HT (10-8M) or to DOI (10-7M) resulted in significant 47 and 42% increases in the number of GR binding sites over control values, respectively (Fig. 1A). There were no significant changes in numbers of GR binding sites after 8-OH-DPAT (10-7M) exposure (Fig. 1A). The specificity of the response to DOI was studied by testing the ability of ketanserin (a 5-HT2 antagonist) to block the effect of the 5-HT2 agonist. The number of GR binding sites in cell cultures treated with DOI (10-7M) in the presence of ketanserin (10-6M) did not significantly differ from controls and decreased significantly compared with cultures exposed to DOI alone (Fig. 1B). Ketanserin alone did not affect GR binding sites (Fig. 1B).

image

Figure 1. Effects of 5-HT, 5-HT receptor agonists and antagonists, and citalopram (CITA) on GR binding sites in hippocampal cell cultures. A: Effects of 5-HT (10-8M), DOI (10-8M), 8-OH-DPAT (DPAT; 10-7M), and CITA (10-7M). B: Effects of ketanserin (KET; 10-6M) and KET with DOI (10-7M). Cultures were exposed to treatments for 4 days starting on day 6 after seeding. Data are mean ± SEM (bars) values, expressed as percentages of the control (C) values, from three experiments. *p < 0.05, significantly different from C values; $p < 0.05, significantly different from DOI values.

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Under the same time schedule as for 5-HT treatment, cells were exposed to the specific 5-HT reuptake inhibitor citalopram (10-7M). This treatment also resulted in a significant increase in GR binding sites (42.5%; Fig. 1A).

GRs in raphe cell cultures

Saturation binding experiments with [3H]dexamethasone at different concentrations (0.2-6 nM) showed specific and saturable binding in raphe cell cultures. Scatchard analysis showed a maximal binding capacity (Bmax) of 117 ± 15 fmol/mg of protein and a binding affinity (KD) of 3.6 ± 1.2 nM (Fig. 2).

image

Figure 2. Saturation curve and Scatchard plot of [3H]dexamethasone (DEX) binding in raphe cells after 10 days in culture. Specific binding is expressed as pmol/mg of protein. B, bound; B/F, bound/free.

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Effect of 5-HT on GR binding sites in raphe cell cultures

For pharmacological experiments, GR binding was measured at a 10 nM saturating concentration of [3H]dexamethasone. Raphe cell cultures were exposed to various concentrations of 5-HT (10-8-10-6M) beginning on day 6 after seeding and for the last 4 days in culture. Exposure to 5-HT (10-7 and 10-8M) induced a significant decrease (-17%) in the number of GR binding sites (Fig. 3A). However, the highest 5-HT concentration tested (10-6M) had no effect on GR binding sites.

image

Figure 3. GR binding sites in raphe cell cultures exposed (A) to 5-HT (10-8-10-6M) and (B) to pargyline (10-5M) or citalopram (10-7M). Cultures were exposed to treatments for 4 days starting on day 6 after seeding. Data are mean ± SEM (bars) values, expressed as percentages of the control (C) values, from two or three experiments. *p < 0.05, significantly different from C values.

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The ability of endogenous 5-HT to decrease the number of GR binding sites was then examined. Under the same time conditions as for 5-HT treatment, cells were exposed to a monoamine oxidase inhibitor, pargyline (10-5M), or to a specific 5-HT reuptake inhibitor, citalopram (10-7M). The number of GR binding sites was significantly decreased in cell cultures following exposure to pargyline or citalopram, dropping 22 and 20% below control values, respectively (Fig. 3B). To ascertain that under our experimental conditions pargyline and citalopram produced an increase in extracellular 5-HT levels, we measured the levels of 5-HT and 5-HIAA in the medium 30, 60, and 120 min after the drug treatments. As soon as 30 min after pargyline application, the levels of 5-HT increased significantly. The maximal effect of pargyline was seen after 120 min, with a 380% increase in 5-HT levels and a 70% decrease in 5-HIAA levels (Fig. 4A). After citalopram treatment the levels of 5-HT increased by 150% at 30 min and peaked at 120 min (428%), whereas 5-HIAA levels started to decrease significantly at 120 min (Fig. 4B).

image

Figure 4. Time course effects of (A) pargyline and (B) citalopram on extracellular levels of 5-HT and 5-HIAA in raphe cell cultures. Data are mean ± SEM (bars) values, expressed as ng/ml/mg of protein, obtained in 12 wells for each group. *p < 0.05, significantly different from control values.

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Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Treatment of raphe cells with the 5-HT1A agonist 8-OH-DPAT at 10-8 or 10-7M for the last 4 days in culture induced significant decreases in number of GR binding sites (-20 and -24%, respectively). However, exposure of the cultures to 10-6M 8-OH-DPAT had no effect on number of GR binding sites (Fig. 5A). Treatment of raphe cells with the 5-HT2 agonist DOI (10-8-10-7M) under the same experimental conditions as for 8-OH-DPAT had no effect on GR binding sites (data not shown). To confirm the involvement of 5-HT1A receptor activation in the observed GR binding site decrease, the ability of WAY 100135 (a 5-HT1A antagonist) to block the effect of 8-OH-DPAT was tested. The numbers of GR binding sites in cell cultures treated with 8-OH-DPAT (10-7M) in the presence of WAY 100135 (10-6M) did not differ from controls and were significantly higher than in cultures exposed to 8-OH-DPAT alone (Fig. 5B). WAY 100135 did not have any intrinsic effect on GR binding sites (Fig. 5B).

image

Figure 5. Effects of 5-HT1A receptor agonist and antagonist on GR binding sites in raphe cell cultures. A: Effects of 8-OH-DPAT (10-8-10-6M). B: Effects of WAY 100135 (WAY; 10-6M) and 8-OH-DPAT (DPAT; 10-7M) with WAY (10-6M). Cultures were exposed to treatments for 4 days starting on day 6 after seeding. Data are mean ± SEM (bars) values, expressed as percentages of the control (C) values, from three experiments. *p < 0.05, significantly different from C values; $p < 0.05, significantly different from DPAT values.

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A time course study of the effects of 8-OH-DPAT (10-8-10-6M) was conducted in which the 5-HT1A agonist was applied for the last 1, 2, 3, 4, and 5 days in culture. Figure 6 shows that the inhibitory effect of 8-OH-DPAT (10-8 and 10-7M) occurred within 2 days of exposure; it was also observed following 3 or 4 days of exposure. By contrast, the number of GR binding sites was unchanged compared with control values when cells were exposed to 10-8 and 10-7M 8-OH-DPAT for 5 days or to the highest dose (10-6M) of the 5-HT1A agonist for 1-5 days (Fig. 6).

image

Figure 6. GR binding sites in raphe cell cultures exposed to 8-OH-DPAT (10-8-10-6M) for the final 1, 2, 3, 4, or 5 days. Data are mean ± SEM (bars) values, expressed as percentages of the control values, from three experiments. The area between the two broken horizontal lines illustrates the range of the SE of the controls. *p < 0.05, significantly different from control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

These data show that 5-HT can regulate GR binding sites in primary cultures of fetal hippocampal and raphe cells. 5-HT induces an increase in the number of GR binding sites in hippocampal cells via the activation of 5-HT2 receptors and a decrease in the number of GR binding sites in raphe cells via the activation of 5-HT1A receptors. Moreover, we show that if the modulation of GR binding sites by citalopram in raphe cells may be mediated by an increase in extracellular 5-HT levels, the effect of citalopram on hippocampal GR binding sites should be mediated by a mechanism that is independent of its effects on synaptic 5-HT levels.

To study the effect of 5-HT on GR binding sites, the choice of using as the experimental model cultures of fetal raphe or hippocampal cells in a serum-free medium was based on the need to control strictly the composition of the extracellular environment, particularly steroid hormone and 5-HT levels. Culturing fetal cells in a serum-free medium that allows for the functional maturation of postmitotic neurons and leads to the death of nonneuronal cells has been shown to result in predominantly neuronal cultures (Faivre-Bauman et al., 1984; Horner et al., 1990).

Under our culture conditions, we found an enhancing effect of 5-HT (10-8M) on the density of hippocampal GR binding sites. This 5-HT effect involved 5-HT2 receptor activation because it was mimicked by the 5-HT2 agonist DOI and the DOI-induced increase in number of GR binding sites could be prevented by the 5-HT2 antagonist ketanserin. DOI shows equal potency at 5-HT2A (previously 5-HT2) and 5-HT2C (previously 5-HT1C) receptors (Hoyer, 1988). This is not surprising, given the structural similarity of these two receptors (Hartig, 1989). However, because ketanserin displays 5-HT2A receptor selectivity (Hoyer et al., 1994), the effect of 5-HT on GR binding sites could preferentially involve 5-HT2A receptor activation. Our results are in line with other studies on hippocampal cell cultures demonstrating a stimulatory effect of 5-HT or 5-HT2 agonist on GR binding sites (Mitchell et al., 1990; Vedder et al., 1993). The necessity of carrying on the treatment for several days before seeing any effect has already been noticed in other in vitro (Mitchell et al., 1990) and in vivo studies. Thus, on the one hand, serotoninergic neuron lesions by 5,7-dihydroxytryptamine have been found to decrease hippocampal GR mRNA levels after several days (Seckl et al., 1990). Similarly, a reduction in GR mRNA levels has been reported following 4 days of 3,4-methyl-enedioxymethamphetamine treatment, which causes selective depletion of 5-HT (Yau et al., 1994). On the other hand, 2 days of p-chloroamphetamine treatment, which, like 3,4-methylenedioxymethamphetamine, leads to the destruction of 5-HT nerve terminals, or 1 day of 3,4-methylenedioxymethamphetamine treatment was found to induce no changes in hippocampal GR binding sites (Lowy et al., 1989; Novotney and Lowy, 1995). In addition, facilitation of serotoninergic neurotransmission after a 14-day treatment with a specific 5-HT reuptake inhibitor like citalopram has also no effect on hippocampal Gr mRNA levels (Seckl and Fink, 1992). The discrepancies between these studies may come from (a) the hormonal status of the animals (intact, adrenalectomized, or adrenalectomized corticosterone-supplemented rats) and (b) the duration of alterations in 5-HT neurotransmission. The use of fetal hippocampal cells cultured in a serum-free medium allows us to suggest that 5-HT acts directly on hippocampal cells to increase the number of Gr binding sites, independently of the presence of corticosterone.

Treatment of hippocampal cells with citalopram leads to an increase in the density of GR binding sites similar to that induced by 5-HT treatment. Because hippocampal cells are devoid of 5-HT-secreting neurons, it appears likely that the effect of citalopram on GR binding sites reported here did not result from an effect of the antidepressant drug on 5-HT uptake. The idea that citalopram exerts its effect independently of any modulation of 5-HT neurotransmission is supported by in vitro and in vivo studies using other antidepressants such as desmethylimipramine, a noradrenaline reuptake inhibitor, and amitriptyline, a mixed noradrenaline/5-HT reuptake inhibitor. Indeed, treatment of primary cultures of rat hypothalamic, amygdala, or cortical neurons with amitriptyline increased GR mRNA levels in these cells (Pepin et al., 1989). Moreover, using a fibroblast cell line that does not produce catecholamines, Pepin et al. (1992) showed that acute desmethylimipramine treatment enhanced GR gene transcription and up-regulated GR numbers. Finally, Rossby et al. (1995) demonstrated that GR mRNA up-regulation occurred in rats treated chronically with desmethylimipramine even after complete neurotoxic lesion of noradrenergic neurons. Gene-specific transcription factors are composed of functionally distinct domains that predispose them to the effects of pharmacological agents (Peterson and Tupy, 1994). Therefore, it is possible, as suggested by Pepin et al. (1992), that the effects of monoamine reuptake inhibitors are exerted at the genomic level via the modulation of transcription factors.

To our knowledge, no reports previously described corticosteroid receptor binding in isolated raphe cells in culture. The finding that GR binding proteins are present in raphe cell cultures is in line with the results of Cintra et al. (1993) showing that GRs are present in the raphe nuclei as early as E15. Bmax and KD values of GR binding sites in cultured raphe cells were similar to those found in binding experiments with adult brainstem tissue from rats adrenalectomized 24 h before they were killed (Sémont et al., 1999). Because strong GR immunoreactivity has been shown in raphe cells containing 5-HT in the neonatal (Cintra et al., 1991) and adult (Harfstrand et al., 1986) rat brain, our results suggest that by E15 GR levels in brainstem are similar to those found in adults.

Our data show that 5-HT at 10-8 to 10-7M decreased the number of GR binding sites in primary cultures of fetal raphe cells. This effect was mimicked by the MAOI pargyline and by the specific 5-HT reuptake inhibitor citalopram, two drugs that induce an increase in extracellular 5-HT content, as previously reported in vivo (Gundlah et al., 1997; Kaehler et al., 1999) and shown under our experimental conditions. These results suggest that a local increase in 5-HT release can regulate GR binding sites in raphe cells. The 5-HT effect involved 5-HT1A receptor activation because it was mimicked by the 5-HT1A receptor agonist 8-OH-DPAT (10-8-10-7M) and the 8-OH-DPAT-induced decrease was prevented by the 5-HT1A receptor antagonist WAY 100135.

The time course study of the 8-OH-DPAT (10-8-10-7M) effect showed that the GR binding site decrease took 2 days to occur. Because the half-life of GR protein is ∼25 h (Dong et al., 1988), this result suggests that the effect of 8-OH-DPAT could involve a decrease in GR protein synthesis. Note that Mitchell et al. (1990) proposed the involvement of GR protein synthesis in the 5-HT-induced increase in the density of GR binding sites in the hippocampus. The inhibitory effect of 8-OH-DPAT was still observed after 3 or 4 days of exposure; however, after 5 days of 8-OH-DPAT treatment, GR binding site values were similar to the control values. These observations suggest that repeated treatment with 8-OH-DPAT for 5 days desensitizes 5-HT1A autoreceptors. Indeed, several electrophysiological studies have demonstrated desensitization of dorsal raphe cells to the 5-HT1A receptor agonist following repeated administration with gepirone, tandospirone, and ipsapirone (Blier and de Montigny, 1987; Schechter et al., 1990; Godbout et al., 1991). More recently, Kreiss and Lucki (1997) reported a diminished ability of 8-OH-DPAT to reduce 5-HT release in the striatum and the hippocampus following repeated treatment with the 5-HT1A agonist. The desensitization of 5-HT1A autoreceptors that develops following chronic drug treatments may only be detectable under appropriate challenge conditions, i.e., those requiring substantial occupation of 5-HT1A autoreceptors. Therefore, it may be hypothesized that in raphe cell cultures these conditions are fulfilled following 5 days of treatment with 10-8 or 10-7M 8-OH-DPAT or following 1 day of treatment with 10-6M 8-OH-DPAT. Accordingly, we showed that GR binding sites were not affected after 1-5 days of treatment with the highest concentration of 8-OH-DPAT.

GRs are present in both neurons and glia (McEwen and Wallach, 1973; McEwen et al., 1986); therefore, the effect of 5-HT on raphe cells could occur in neurons as well as in glial cells. Nevertheless, considering that the 5-HT1A receptors were found almost exclusively in the somatodendritic compartment of the neurons and were very rarely observed in processes belonging to glial cells (Kia et al., 1996) and that cultures in a serum-free medium resulted in predominantly neuronal cultures, we may assume that the observed 5-HT effect occurs on GR binding sites located in neurons.

Changes in corticosteroid concentration and serotoninergic neurotransmission are present in anxiety disorders, on which 5-HT1A receptor agonists are clinically effective (Deakin, 1993). Via the activation of GRs by corticosterone, stressful stimuli enhance the activity of tryptophan hydroxylase (Boadle-Biber et al., 1989) and increase brain 5-HT turnover (Clement et al., 1993) and extracellular 5-HT levels (Shimizu et al., 1992). Reduction of 5-HT neurotransmission is thought to have an anxiolytic effect (Sommermeyer et al., 1993). The role of presynaptic 5-HT1A receptors located in the raphe nuclei in mediating the anxiolytic effects of 5-HT1A agonists has been demonstrated in animal models (Boadle-Biber et al., 1989). It has been suggested that the anxiolytic effect of 5-HT1A receptor agonists requires action in the dorsal raphe nucleus through the stimulation of somatodendritic 5-HT1A autoreceptors, resulting in less firing of serotoninergic neurons and a subsequent reduction in 5-HT release (Sommermeyer et al., 1993). Moreover, because GR antagonists like RU 38486 were shown to display anxiolytic-like activity in rats (Korte et al., 1995), possibly through an increase in 5-HT autoinhibition in the raphe (Laaris et al., 1995), it has been hypothesized that attenuation of GR-mediated effects reduces anxiety. GR activation in the raphe nuclei reduces the efficiency of the negative control of the serotoninergic neurons’ electrical activity, owing to the stimulation of somatodendritic 5-HT1A autoreceptors (Laaris et al., 1995). Thus, it can be hypothesized that the decreased density of GR binding sites in the raphe nuclei following 5-HT1A receptor activation contributes to the anxiolytic action of 5-HT1A agonists by restoring the efficiency of 5-HT1A autoreceptors in the negative control of the electrical activity of serotoninergic neurons.

In conclusion, our data showed for the first time an inhibitory effect of 5-HT on GR binding sites in fetal raphe nuclei. They also indicate that during fetal life, the regulation of GR binding sites by 5-HT is structure-dependent and involves the activation of different 5-HT receptor subtypes. Furthermore, this study suggests that specific 5-HT reuptake inhibitors might modulate GR expression in central structures devoid of serotoninergic neurons via a mechanism that is independent of their capacity to block 5-HT reuptake.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

This work was supported by INSERM and the Université de la Méditerranée. We are grateful to Valérie Kogalama and Mireille Seguin for their secretarial assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations
  5. RESULTS
  6. Effect of 8-OH-DPAT on GR binding sites in raphe cell cultures
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES
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