Anatomical and biochemical evidence for the synthesis of unconjugated and sulfated neurosteroids in amphibians

Brain Research Reviews 37 (2001) 13–24 www.elsevier.com / locate / bres

Review

Anatomical and biochemical evidence for the synthesis of unconjugated and sulfated neurosteroids in amphibians a, a b b Ayikoe G. Mensah-Nyagan *, Delphine Beaujean , Van Luu-The , Georges Pelletier , a Hubert Vaudry a

European Institute for Peptide Research ( IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Sante´ et de ´ ´ au Centre National de la Recherche Scientifique ( UA CNRS), University of Rouen, la Recherche Medicale ( INSERM U-413), Unite´ Affiliee 76821 Mont-Saint-Aignan, France b MRC Group in Molecular Endocrinology, Laval University Hospital Center, Quebec, Canada G1 V 4 G2 Accepted 12 June 2001

Abstract Various studies have shown that, in mammals, neurons and glial cells are capable of synthesizing bioactive steroids, or neurosteroids, which regulate the activity of the central nervous system (CNS). However, although steroid hormones are involved in the regulation of behavioral and neuroendocrine processes in amphibians, neurosteroid biosynthesis has never been studied in the CNS of non-mammalian vertebrates. Reviewed here are several data sets concerning the production of unconjugated and sulfated neurosteroids in amphibians. These data were obtained by investigating the immunohistochemical localization and activity of 3b-hydroxysteroid dehydrogenase (3b-HSD), 17b-hydroxysteroid dehydrogenase (17b-HSD) and hydroxysteroid sulfotransferase (HST), in the frog brain. Numerous 3b-HSD-immunoreactive neurons were detected in the anterior preoptic area, nucleus of the periventricular organ, posterior tuberculum, ventral and dorsal hypothalamic nuclei. 17b-HSD-like immunoreactivity was found in ependymal gliocytes bordering the lateral ventricles of the telencephalon. Two populations of HST-immunoreactive neurons were localized in the anterior preoptic area and the dorsal magnocellular nucleus of the hypothalamus. High amounts of progesterone (PROG), 17-hydroxyprogesterone (17OH-PROG), testosterone (T) and dehydroepiandrosterone sulfate (DHEAS) were measured in the frog brain by combining HPLC analysis of tissue extracts with radioimmunoassay detection. Incubation of telencephalic or hypothalamic explants with tritiated pregnenolone ([ 3 H]PREG) yielded the synthesis of various metabolites including PROG, 17OH-PROG, DHEA and T. Incorporation of [ 35 S]39-phosphoadenosine 59-phosphosulfate ([ 35 S]PAPS) and [ 3 H]PREG or [ 3 H]DHEA into frog brain homogenates led to the formation of [ 3 H,35 S]pregnenolone sulfate ([ 3 H,35 S]PREGS) or [ 3 H,35 S]DHEAS, respectively. Altogether, these results demonstrate that the process of neurosteroid biosynthesis occurs in amphibians as previously seen in mammals.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Amphibian; Hydroxysteroid sulfotransferase; 3b-Hydroxysteroid dehydrogenase; 17b-Hydroxysteroid dehydrogenase; Immunocytochemistry; Neurosteroid

Contents 1. Introduction ............................................................................................................................................................................................ 2. Anatomical and cellular distribution of 3b-HSD in the frog brain................................................................................................................ 3. Anatomical and cellular distribution of 17b-HSD in the frog brain ..............................................................................................................

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* Corresponding author. Present address: Laboratoire de Neurophysiologie Cellulaire et Integree, ´ Centre National de la Recherche Scientifique (CNRS), Unite´ Mixte de Recherche 7519, Universite´ Louis Pasteur, 21, rue Rene´ Descartes, 67084 Strasbourg, France. Tel.: 133-3-9024-1451; fax: 133-3-88613347. E-mail address: [email protected] (A.G. Mensah-Nyagan). 0165-0173 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0165-0173( 01 )00110-2

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4. Anatomical and cellular distribution of HST in the frog brain ..................................................................................................................... 5. In vivo evidence for the biosynthesis of steroids in the CNS of amphibians ................................................................................................. 5.1. Unconjugated neurosteroids ............................................................................................................................................................. 5.2. Sulfated neurosteroids ..................................................................................................................................................................... 6. In vitro evidence for the biosynthesis of steroids in the CNS of amphibians ................................................................................................. 6.1. Unconjugated neurosteroids ............................................................................................................................................................. 6.2. Sulfated neurosteroids ..................................................................................................................................................................... 7. Conclusion and physiological implications ................................................................................................................................................ Acknowledgements ...................................................................................................................................................................................... References...................................................................................................................................................................................................

1. Introduction During the two last decades, several studies have demonstrated that, in mammals, nerve cells are capable of synthesizing various biologically active steroids, or neurosteroids, which along with steroid hormones produced by the adrenal gland and gonads, regulate brain activity (for review, see Ref. [11]). Pharamacological and behavioral investigations have shown that neurosteroids are involved in the control of important neurophysiological mechanisms [64,104,105]. For instance, infusion of pregnenolone sulfate (PREGS) into the rat nucleus basalis stimulates learning and it has been observed that deficient cognitive performance in aged rodents depends on low PREGS levels in the hippocampus [71,115]. Allopregnanolone, which attenuates the hormonal responses to stress, also exerts glucocorticoid-like effects on vasopressin gene transcription in the rat hypothalamus, suggesting that neurosteroids may participate in the regulation of endocrine system activity [91]. The dosedependent effects of PREGS on locomotion of mice placed in a novel environment indicate the existence of a possible role of neurosteroids in adaptation to novelty [33]. It has also been demonstrated that dehydroepiandrosterone (DHEA) and methyl-DHEA are capable of inhibiting aggressiveness in rodents by decreasing the cerebral concentration of PREGS [44,103,121]. Moreover, in addition to their effects on the activity of the central nervous system (CNS), neurosteroids have been shown to also regulate myelin formation and regeneration of peripheral nerves [50]. There is now increasing evidence indicating that, besides their actions at the transcriptional level [72], neuroactive steroids may act on nerve cells via membrane receptors including proper membrane receptors [87,96], GABAA [64,94], NMDA [16,120] and sigma [78] receptors. Although the involvement of neurosteroids in the regulation of neurophysiological processes has been shown in mammals, the biochemical pathways leading to the synthesis of steroids in the nervous system have long remained incompletely identified [11,17]. Moreover, in spite of the crucial role played by steroid hormones in the regulation of behavioral and neuroendocrine mechanisms

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in fish [49,122], amphibians [79,80] and birds [9,88], the biosynthesis of neurosteroids had never been studied in the nervous system of non-mammalian vertebrates. In order to investigate the occurrence of neurosteroidogenesis in amphibians, the immunohistochemical localization and biological activity of three key steroidogenic enzymes were studied in the CNS of the European green frog, Rana ridibunda. These enzymes are 3b-hydroxysteroid dehydrogenase or 3b-HSD, 17b-hydroxysteroid dehydrogenase or 17b-HSD and hydroxysteroid sulfotransferase or hydroxysteroid sulfotransferase (HST) (for review, see Ref. [76]).

2. Anatomical and cellular distribution of 3b-HSD in the frog brain The enzyme 3b-HSD plays a pivotal role in the biosynthesis of all classes of steroid hormones by catalyzing the conversion of D5 -3b-hydroxysteroids such as pregnenolone (PREG), 17-hydroxypregnenolone (17OHPREG) and DHEA into D4 -3-ketosteroids including progesterone (PROG), 17-hydroxyprogesterone (17OHPROG) and androstenedione, respectively. This enzyme, which was originally identified in steroid hormone-producing organs, has also been found in other tissues including prostate, breast, liver, kidney and skin [52]. Molecular cloning of cDNAs encoding 3b-HSD has revealed the existence of two and six isoforms of the enzyme in human [61,101] and rodents [1,69,108,123], respectively. The molecular characterization of 3b-HSD cDNA has not yet been investigated in amphibians. However, two isoforms of cDNA encoding for 3b-HSD have been cloned in fish and their deduced amino acid sequences both showed 53% similarities with mouse 3b-HSD isoenzymes [55]. In addition, the 3b-HSD cDNA isolated from a chicken adrenal gland cDNA library encodes for a 377 amino acid sequence which shares 54–57% overall identity with 3bHSD isoforms of human, macaque, bovine, mouse rat and rainbow trout [82]. Collectively, these data suggest that the 3b-HSD gene may be highly conserved during vertebrate evolution. The first evidence indicating the occurrence of 3b-HSD activity in the CNS has been provided in mammals by

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biochemical approaches [10,45,102,119]. However, the anatomical and cellular localization of 3b-HSD in the mammalian brain remained unknown until recently [11]. The first immunohistochemical mapping of 3b-HSD in the CNS has been performed in the frog R. ridibunda by using an antiserum against type I human placental 3b-HSD [73]. The immunoreactivity was found in several populations of neurons located in the anterior preoptic area, nucleus of the periventricular organ, posterior tuberculum, suprachiasmatic nucleus and the dorsal and ventral hypothalamic nuclei (Fig. 1). The 3b-HSD-immunoreactive material had a granular aspect and was apparently sequestered in organelles located in cytoplasm and cytoplasmic extensions (Fig. 2A). A dense network of 3b-HSD-positive beaded nerve fibers was observed throughout the telencephalon, diencephalon and mesencephalon [23,73]. The antiserum used to detect 3b-HSD in the frog brain has been successfully applied to the immunocytochemical localization of the enzyme in classical steroid-producing organs of mammals such as the adrenal, testis, ovary and placenta [28–30]. The specificity of the immunostaining observed in the CNS of frog has been confirmed by preabsorption experiments with purified type I human placental 3b-HSD [23,73]. However, although the antibodies were raised against type I human 3b-HSD [61],

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they also recognize other isoforms including type II 3bHSD which is predominantly expressed in the adrenal gland and gonads [28–30,54]. Therefore, the immunoreactive material detected in the frog brain may correspond to any variant of the 3b-HSD family. In situ hybridization studies have shown that, in the CNS of rat, the 3b-HSD gene is also expressed in neuronal populations located in several nuclei throughout the telencephalon, diencephalon and mesencephalon, particularly in the olfactory bulb, nucleus accumbens, hippocampus, thalamus and hypothalamus [42]. However, 3b-HSD mRNAs were found in various neurons of the area of medulla bordering the rat fourth ventricle [32] and in Purkinje neurons of the quail cerebellum [37,114], while, in the frog, these brain regions were devoid of 3b-HSD-like immunoreactivity (Fig. 1). These data suggest the existence of variations in the anatomical distribution of 3b-HSD in the CNS of vertebrate species.

3. Anatomical and cellular distribution of 17b-HSD in the frog brain The interconversion of 17-ketosteroids (androstenedione and estrone) and 17b-hydroxysteroids (testosterone (T)

Fig. 1. Schematic parasagittal section depicting the distribution of 3b-HSD- (orange), 17b-HSD- (red) and HST- (green) immunoreactive cell bodies (stars) and fibers or processes (dots) in the frog brain. The density of the symbols is meant to be proportional to the relative abundance of the immunoreactive elements. The anatomical structures are designated according to the nomenclature of Northcutt and Kicliter [85] and Neary and Northcutt [83]. CGL, corpus geniculatus lateralis; HYP, hypothalamus; Mdg, dorsal magnocellular nucleus; ME, median eminence; NDB, nucleus of the diagonal band of Broca; NPv, nucleus of the periventricular organ; OC, optic chiasma; OLF. BULB, olfactory bulb; OT, optic tract; Pdis, hypophysis, pars distalis; PI, hypophysis, pars intermedia; PN, hypophysis, pars nervosa; Poa, anterior preoptic area; Rhombenc, rhombencephalon; SC, suprachiasmatic nucleus; Teg, tegmentum; TP, posterior tuberculum.

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and 17b-estradiol) is catalyzed by 17b-HSD. This enzyme thus plays a crucial role in the biosynthesis and inactivation of sex steroid hormones. Molecular cloning of 17bHSD cDNAs and biochemical characterization have revealed the existence of seven isoforms designated types I–VII [7,8,13,14,84]. The types I, III and V 17b-HSD catalyze mainly reductive reactions leading to the formation of T and 17b-estradiol. The isoenzymes type II and IV are preferentially involved in oxidative reactions which are responsible for androstenedione and estrone synthesis [7]. The type VI 17b-HSD mainly converts 5a-androstane-3a, 17b-diol to androsterone and type VII, which catalyzes the transformation of estrone to estradiol, shares 89% sequence identity with prolactin receptor-associated protein [26,27,84]. Until now, only one 17b-HSD isoenzyme has been characterized in non-mammalian vertebrates. The isoforms 17b-HSD type I cloned in fish [46] and birds [118] share 50% overall identity with mammalian types I 17b-HSD isoenzymes. The different forms of 17b-HSD were detected in various peripheral steroidogenic tissues in inframammalian vertebrates [46,118] as well as in mammals [2,15,38,53,66]. The occurrence of 17b-HSD activity in the CNS has been shown in mammals several years ago [98,100] but the immunohistochemical localization of the enzyme in the brain is recent [92]. The anatomical distribution of 17bHSD was investigated in the CNS of frog by using an antibody against type I human placental 17b-HSD [74,75]. Glial cells exhibiting 17b-HSD-like immunoreactivity were observed in the telencephalon and the rostral part of diencephalon (Fig. 1). A dense population of 17b-HSDpositive ependymal cells was found within the periventricular zone of the medial pallium (Fig. 2B). Another group of 17b-HSD-immunoreactive glial cells was also visualized in the periventricular area of the lateral septum (Fig. 1). The ependymal glial cells containing 17b-HSDimmunoreactive material projected their linear processes towards the rostral part of diencephalon (Fig. 2A). Various networks of 17b-HSD-positive processes were detected in the anterior preoptic area, corpus geniculatus lateralis and thalamus (Fig. 1). The antiserum used for the determination of the anatomical and cellular distribution of 17bHSD in the frog brain has also been applied successfully to localize the enzyme in the human placenta [31] and the rat brain [92]. In the CNS of rodents, 17b-HSD-like immuno-

Fig. 2. Confocal laser scanning microscope photomicrographs of 3bHSD- (orange), 17b-HSD- (red) and HST- (green) immunoreactive cells in the frog brain. (A) Frontal section through the rostral diencephalon showing the presence of 3b-HSD-immunoreactive neurons in the anterior preoptic area (Poa). (B) Frontal section through the telencephalon showing 17b-HSD-immunoreactive ependymal cells bordering the lateral ventricle (V) of the medial pallium (MP). (C) Frontal section through the hypothalamus showing the presence of HST-immunoreactive neurons in the dorsal magnocellular nucleus (Mgd). III, third ventricle. Scale bars represent 10 mm.

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reactivity was found in ependymal cells and astrocytes of the hippocampus, cerebral cortex, thalamus and hypothalamus [92]. These data indicate that, in the brain of both amphibians and mammals, type I 17b-HSD is exclusively expressed in glial cells. However, 17b-HSD-immunoreactive cells have been detected in the telencephalon, diencephalon and mesencephalon of rat, while, in the frog brain, these cells were only observed in the telencephalon [74,75,92]. These results suggest the existence, among vertebrates, of species differences in the anatomical distribution of 17b-HSD in the CNS.

4. Anatomical and cellular distribution of HST in the frog brain Sulfotransferases are a family of enzymes responsible for the conjugation of a wide range of molecules using 39-phosphoadenosine 59-phosphosulfate (PAPS) as the donor of sulfate. A member of this family is HST which catalyzes the biosynthesis of sulfated steroids by transferring the sulfonate moiety from PAPS on the 3-hydroxyacceptor site of steroid substrates. The enzyme HST, which has been cloned in classical steroidogenic organs including the adrenal and gonads, was also characterized in other tissues such as the liver, kidney and jejunum (for review, see Ref. [111]). Several years ago, sulfoconjugation of DHEA has been shown in the primate brain tissue in vivo and in vitro, suggesting the occurrence of HST activity in the CNS of mammals [47,48]. In addition, high amounts of PREGS and dehydroepiandrosterone sulfate (DHEAS) have been detected in the rat brain and the castration and adrenalectomy of the animals did not modify the cerebral concentrations of sulfated steroids [18,19]. The existence of HST bioactivity in the rodent brain has also been demonstrated by in vitro studies [95]. However, the cellular distribution of HST in the CNS had long remained unknown since immunohistochemical attempts to localize the enzyme in the human and rat brain have been unsuccessful [89,107]. The availability of an antiserum raised against the rat liver HST made it possible to determine the distribution of HST-like immunoreactive structures in the CNS of frog [12]. Two populations of HST-positive neurons were localized in the anterior preoptic area and the dorsal magnocellular nucleus of the hypothalamus (Fig. 1). The HST-immunoreactive material was found in cytoplasm and cytoplasmic extensions (Fig. 2C). All nerve processes containing HST-like immunoreactivity exhibited the typical varicose aspect of beaded nerve fibers [12]. A dense bundle of HST-positive fibers was visualized in the ventral hemispheric zone. These fibers, which were radially orientated towards the nucleus accumbens, crossed the medial septum and the nucleus of the diagonal band of Broca (Fig. 1). Numerous HST-immunoreactive fibers were also observed in the corpus geniculatum thalamicum, posterior

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thalamic nucleus, basal optic nucleus and nucleus reticularis isthmi. The specificity of HST-immunostaining detected in the frog brain has been established by preabsorption experiments with the synthetic peptide hapten corresponding to the sequence 180–192 of rat liver HST [12,86]. However, the 13 amino acid peptide used for raising antibodies is located in a domain that is highly conserved among the various isoforms of HST cloned in mammalian species [111]. Therefore, the immunoreactive material detected in the CNS of frog may correspond to isoenzyme(s) of either the 3a-HST family, which are mainly selective for 3a-hydroxysteroids [59], or the 3bHST family, also referred to act as DHEA-HST [34,63] or PREG-HST [40,62]. In agreement with this hypothesis, a recent study of the cellular distribution of DHEA-HST in the rat brain has revealed that this HST isoform is also expressed by neuronal populations located in the midbrain [4].

5. In vivo evidence for the biosynthesis of steroids in the CNS of amphibians The occurrence of endogenous production of unconjugated and sulfated steroids in the CNS of frog has been investigated by combining high performance liquid chromatographic (HPLC) analysis of brain tissue extracts with radioimmunoassay detection (for review, see Ref. [76]).

5.1. Unconjugated neurosteroids High amounts of endogenous D4 -3-ketosteroids such as PROG and 17OH-PROG were measured in the telencephalon and hypothalamus of the frog (Table 1). The concentration of PROG in the frog hypothalamus was only four times lower than that detected in the adrenal gland which is the main source of plasma PROG in male frogs [58]. It has been demonstrated that, in male rats, the concentrations of PROG in brain and plasma are in the same range [20]. In contrast, the level of PROG measured in the frog brain was 100 times higher than in plasma, while 17OH-PROG was not detectable in frog blood (Table 1). Therefore, these results suggest that PROG and 17OH-PROG are produced, in vivo, in the CNS of frog. However, the possibility that a proportion of the brain D4 -3-ketosteroids could originate from selective uptake of steroids from plasma should not be excluded. The combination of HPLC analysis and radioimmunoassay detection also showed the occurrence, in the telencephalon and diencephalon of both female and male frogs, of substantial amounts of 17b-hydroxysteroids including T and dihydrotestosterone or 5a-DHT [74,75]. The cerebral concentrations of T and 5a-DHT are not modified by castration in male frogs (Table 1), suggesting that these two 17b-hydroxysteroids detected in the forebrain of amphibians do not originate from the testis. The chemical

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Table 1 Concentration of endogenous PROG, 17OH-PROG, T, 5a-DHT and DHEAS detected in the brain and blood of frog Tissue

Steroids Unconjugated D4 -3-ketosteroids

Telencephalon Hypothalamus Blood

Unconjugated 17b-hydroxysteroids

Sulfated 3-hydroxy-steroid

PROG (pg/mg tissue or ml blood)

17OH-PROG (pg/mg tissue or ml blood)

T (pg/mg tissue or ml blood)

5a-DHT (pg/mg tissue or ml blood)

Male

Male

Female

Male

Castrated male

Female

Male

Castrated male

Male

150620 140627 1.060.5

5567 4567 Undetectable

4.260.3 2.760.2 0.0260.01

4.060.03 2.760.01 0.0360.01

3.760.2 1.760.1 Undetectable

4.060.3 1.860.1 0.0260.01

3.760.02 1.760.1 0.0360.01

3.360.2 1.660.1 Undetectable

4.3460.2 13.4560.2 1.1760.08

structure of the endogenous T extracted from the frog brain has been clearly confirmed by the combination of HPLC analysis with gas chromatography-mass spectrometry identification [74,75]. This approach revealed that the endogenous steroid and synthetic T had the same retention time on the gas chromatograms and the mass spectra of the two steroids were similar.

5.2. Sulfated neurosteroids The presence of substantial amounts of DHEAS-immunoreactive material, which coeluted in the HPLC system with synthetic DHEAS, has been demonstrated in the CNS of frog [77]. The concentrations of DHEAS measured in the frog telencephalon and hypothalamus were four and 11 times higher than in plasma (Table 1). As sulfate ester of steroids are hydrophilic compounds which hardly cross the blood–brain barrier [47,48], it appears that the endogenous DHEAS detected in the CNS of frog cannot originate from selective uptake from the circulating DHEAS pool but is actually synthesized in situ. Moreover, a study conducted in the frog Rana nigromaculata has revealed the occurrence of high concentrations of PREGS in the brain of both female and male animals during the breeding and post-breeding seasons, independent of the plasma steroid level [112]. Collectively, these data provide evidence for the production, in vivo, of sulfated neurosteroids in amphibians.

6. In vitro evidence for the biosynthesis of steroids in the CNS of amphibians

6.1. Unconjugated neurosteroids The synthesis of unconjugated D4 -3-keto- and 17bhydroxy-neurosteroids was investigated by using the pulse-

DHEAS (pg/mg tissue or ml blood)

chase technique with [ 3 H]PREG as a precursor. In the hypothalamus, the formation of various [ 3 H]D4 -3-ketosteroids was observed, including [ 3 H]PROG and [ 3 H]17OHPROG [73]. Trilostane, a specific inhibitor of 3b-HSD [51], significantly reduced the conversion of [ 3 H]PREG into [ 3 H]17OH-PROG. Incubation of telencephalic explants with [ 3 H]PREG yielded the synthesis of [ 3 H]D4 -3ketosteroids (PROG, 17OH-PROG and androstenedione), [ 3 H]D5 -3b-hydroxysteroids (DHEA and 17OH-PREG) and [ 3 H]17b-hydroxysteroids (T and 5a-DHT), in female as well as in male frogs [74,75]. The frog rhombencephalic tissue was used as control for these pulse-chase experiments as this area of the brain was totally devoid of 3b-HSD- and 17b-HSD-immunoreactivities. In this case, only one oxidative derivative of [ 3 H]PREG was synthesized [73–75]. Kinetic studies indicated that the biosynthetic pathways leading to the formation of androgens in the frog telencephalon are similar to that occurring in Leydig cells (Fig. 3).

6.2. Sulfated neurosteroids A double labeling pulse-chase approach using both [ 3 H]-labeled steroid precursors ([ 3 H]PREG or [ 3 H]DHEA) and a 35 S-labeled sulfate donor ([ 35 S]PAPS) was developed to investigate, in vitro, the biosynthesis of steroid sulfates [12]. Incubation of frog telencephalic and hypothalamic homogenates with [ 35 S]PAPS and [ 3 H]PREG yielded the formation of several [ 3 H,35 S]-labeled compounds including PREGS and testosterone sulfate (TS). When [ 3 H]DHEA and [ 35 S]PAPS were used as precursors, one of the [ 3 H,35 S]-labeled metabolites coeluted with DHEAS [12]. Neosynthesis of [ 3 H]PREGS and [ 3 H]DHEAS was reduced by 2,4-dichloro-6-nitrophenol, a specific inhibitor of HST [99]. No formation of sulfated steroids was observed with the rhombencephalon which

Fig. 3. Biochemical pathways of unconjugated and sulfated steroid synthesis in the frog brain. 3b-HSD, 3b-hydroxysteroid dehydrogenase /D5 -D4 isomerase; 17b-HSD, 3b-hydroxysteroid dehydrogenase; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; HST, hydroxysteroid sulfotransferase; P450c 17 , cytochrome P450c 17 (complex 17a-hydroxylase / 17, 20 lyase); P450scc, cytochrome P450 side chain cleavage; PREG, pregnenolone; PREGS; pregnenolone sulfate.

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did not contain HST-immunoreactive elements [12,77]. These results demonstrate that the HST-like immunoreactivity in the frog telencephalon and hypothalamus actually corresponds to an active form of the enzyme which may be responsible for the production of sulfated 3-hydroxy-neurosteroids in the amphibian brain through a pathway similar to that observed in mammals (Fig. 3).

7. Conclusion and physiological implications The neuronal populations containing 3b-HSD, HSTimmunoreactive neurons and several 17b-HSD-positive processes are found in various nuclei of the frog diencephalon, such as the anterior preoptic area, the magnocellular nucleus, and the dorsal and ventral hypothalamic nuclei [76]. These brain areas, which also contain the neuronal systems involved in the regulation of the activity of pituitary cells, are generally considered as the frog hypothalamic hypophysiotropic centers (for review, see Ref. [6]). Therefore, it appears that neurosteroids may exert neuroendocrine functions by modulating the biosynthesis and / or release of hypophysiotropic neurohormones. Moreover, the frog diencephalic regions, where unconjugated steroid-secreting neurons (3b-HSD) and sulfated steroid-producing cells (HST) are located, also receive afferent fibers expressing various neurotransmitters and neuropeptides including catecholamines [39], GABA [36], corticotropin-releasing hormone [113], vasotocin [56,116], neuropeptide Y [21] and diazepam-binding inhibitor or DBI [65]. These data suggest that the biosynthesis of unconjugated and sulfated neurosteroids may be regulated by classical neurotransmitters and / or neuropeptides. Since GABAA receptors are borne by several cell bodies located in the frog diencephalon [5], and as most of the actions of neurosteroids appear to be mediated through these receptors [64,94], the possible feed-back regulation of neurosteroidogenesis by GABA was recently investigated in amphibians [24]. This study, which indicates that steroid-producing neurons in the frog hypothalamus express GABAA receptor a 3 and b 2 / b 3 subunits, demonstrates that GABA inhibits the activity of 3b-HSD in the CNS through activation of GABAA receptors. It has also been shown that the two endozepines or DBI processing fragments, triakontatetraneuropeptide DBI [17–50] and octadecaneuropeptide DBI [33–50], both stimulate the production of neurosteroids in the frog brain by acting, respectively, via the peripheral-type and the central-type benzodiazepine receptors [23,25]. Collectively, these data suggest that various neurotransmitters and neuropeptides may induce some of their neurophysiological effects through modulation of neurosteroid biosynthesis. In support of this hypothesis, it has been demonstrated that neuropeptides such as endozepines and neuropeptide Y are involved in the regulation of behavioral activities including

anxiety [22,41,43,81,90] and feeding [70,106,109,110] that are also modulated by neurosteroids [3,64,93,97]. It is well established that the limbic system of vertebrates is involved in the regulation of various behavioral processes. In amphibians, the limbic system is located in the telencephalon, particularly in the septal area and the pallium, where 17b-HSD-immunoreactive glial cells have been localized [74,75,83,85,117]. These data suggest that T produced in the telencephalon by 17b-HSD-containing cells may act locally to control behavioral activities. Whether the endogenous T of the CNS may directly interact with androgen receptors or whether it requires aromatization to form estradiol [60,67,88] deserves further investigation. In this respect, it should be noted that TS is one of the sulfated steroids synthesized from [ 3 H]PREG by telencephalic and hypothalamic homogenates [12]. Therefore, sulfation of T could be an additional possibility to 5a-reduction [68] and aromatization [60,88] which may be required for the expression of its behavioral effects. Sulfate ester of 3-hydroxysteroids such as PREGS and DHEAS are potent regulators of neuronal activity in mammals [11,35,64]. The occurrence of the biosynthesis of sulfate ester of PREG and DHEA in the CNS of frog, in vivo [77,112] and in vitro [12], strongly suggests that sulfated neurosteroids may also control various neurophysiological processes in amphibians as previously described in mammals [11]. In agreement with this hypothesis, electrophysiological studies have shown the existence of potent modulatory effects of neuroactive steroid sulfates on the GABAA receptor activity in frog pituitary melanotrope cells [57]. Moreover, the good correlation observed during the annual cycle between high cerebral PREGS concentrations and intense activity periods in amphibians [112], indicates that sulfated neurosteroids may be involved in adaptive mechanisms. Finally, the following evidence obtained in frog, i.e. (i) the presence of key steroidogenic enzymes in the brain [76,112], (ii) the occurrence of unconjugated and sulfated steroid biosynthesis in the CNS [12,73–77,112], and (iii) the identification of neuroanatomical and functional relationship between the GABAergic system, endozepineproducing cells and neurosteroid-secreting neurons [23– 25], indicates that the amphibian brain is a suitable model in which to investigate the process of neurosteroidogenesis and its physiological significance.

Acknowledgements This work was supported by grants from the INSERM ` des Affaires Etrangeres ` (U 413), the Ministere (France´ Quebec exchange program no. PV-P-73-9), and the Conseil ´ Regional de Haute-Normandie. D.B. was the recipient of a ` de l’Education Nationale, de fellowship from the Ministere ´ l’Enseignement Superieur et de la Recherche.

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