NEUROSTEROIDS: A NOVEL FUNCTION OF THE BRAIN

Psychoneuroendocrinology, Vol. 23, No. 8, pp. 963 – 987, 1998 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0306-4530/98 $ - see front matter

PII: S0306-4530(98)00071-7

NEUROSTEROIDS: A NOVEL FUNCTION OF THE BRAIN E.E. Baulieu INSERM U 488, 80 rue du Ge´ne´ral Leclerc, 94276 Le Kremlin-Biceˆtre Cedex, France

SUMMARY Neurosteroids are synthetized in the central and peripheral nervous system, particularly but not exclusively in myelinating glial cells, from cholesterol or steroidal precursors imported from peripheral sources. They include 3-hydroxy-D5-compounds, such as pregnenolone (PREG) and dehydroepiandrosterone (DHEA), their sulfates, and reduced metabolites such as the tetrahydroderivative of progesterone 3a-hydroxy-5a-pregnane-20-one (3a,5a-TH PROG). These compounds can act as allosteric modulators of neurotransmitter receptors, such as GABAA, NMDA and sigma receptors. Progesterone (PROG) is also a neurosteroid, and a progesterone receptor (PROG-R) has been identified in peripheral and central glial cells. At different places in the brain, neurosteroid concentrations vary according to environmental and behavioral circumstances, such as stress, sex recognition and aggressiveness. A physiological function of neurosteroids in the central nervous system is strongly suggested by the role of hippocampal PREGS with respect to memory, observed in aging rats. In the peripheral nervous system, a role for PROG synthesized in Schwann cells has been demonstrated in the repair of myelin after cryolesion of the sciatic nerve in vivo and in cultures of dorsal root ganglia neurites. It may be important to study the effect of abnormal neurosteroid concentrations/metabolism with a view to the possible treatment of functional and trophic disturbances of the nervous system. © 1998 Elsevier Science Ltd. All rights reserved. Keywords—DHEA, dehydroepiandrosterone; PREG, pregnenolone; PROG, progesterone; GABAA receptor; Memory; Myelination.

NEUROSTEROIDS: THE BEGINNING The work to describe the synthesis and metabolic pathways of neurosteroids, and establish their physiological and pathological function and mechanism(s) of action has encountered some major difficulties: (1) We (see acknowledgements) met many analytical problems, qualitative and quantitative because of the low concentration and the lipoı¨dal nature of neurosteroids, which have to be separated from the highly lipidic constituents of neural tissues. Strictly controlled conditions had to be established since neurosteroid concentrations vary according to the time of the day, the lighting schedule, the food, the presence of other animals, the habituation to handling, etc…; (2) The overall dynamics of the synthesis of neurosteroids is unknown, since their turnover cannot be determined and no appropriate techniques are available for describing their compartmentation; (3) Quantitative aspects are particularly difficult to master since many neurosteroids are also secreted by peripheral glands, may cross the BBB and also easily attain peripheral nerves, Address correspondence and reprint requests to: E.E. Baulieu, INSERM U 488, 80 rue du Ge´ne´ral Leclerc, 94276 Le Kremlin-Biceˆtre Cedex, France (Tel: 33 1 49591882; Fax: 33 1 45211940; E-mail: [email protected]). 963

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eventually mixing with neurosteroids. In fact, the respective distribution and contribution of steroids imported into the nervous system and of those synthetized in situ remain difficult to assess and consequently, we do not necessarily know what the respective targets are; and (4) Binding studies and thus the search for receptors are especially hard in a lipid rich milieu, as often encountered in work with nervous tissue and its membranes, again because of the liposolubility of steroids. We essentially followed two strategic lines. The first consisted in establishing the synthesis and metabolic pathways of steroids in the nervous system (Fig. 1), including the characterization of the corresponding enzymes and receptors. The second series of studies was to determine changes and, if possible, function of neurosteroids under various physiological or pathological conditions. In initial experiments, we measured steroids remaining in the brain after removal of potential glandular sources of steroids, that is after adrenalectomy and gonadectomy (Table I). In rats, we essentially noted the persistency of DHEA and its conjugates after several weeks, and the decrease of PREG and its conjugates, though this was only partial, as if the ablation of endocrine glands had led to the suppression of only the imported steroid (PREG is a circulating steroid in the rat, and is also synthesized in the brain). In male rats, after operation, we observed the persistency of a low but easily measured level of PROG while, in contrast, testosterone and testosterone sulfate disappeared rapidly from the brain tissue (Corpe´chot et al., 1981, 1983, 1993). Consistent with the hypothesis that the persistency of steroids after adrenalectomy-gonadectomy is not due to retention of compounds originally circulating in the blood, we observed the rapid release of uptaken radioactive DHEA and PREG following their peripheral administration (Corpe´chot et al., 1983). A circadian cycle of brain PREG and DHEA, unrelated to blood steroid levels, was also observed (Robel et al., 1986; Synguelakis et al., 1985), and brain PREG was found to be high for several days after birth in rats even when the adrenal steroid output is low (Robel and Baulieu, 1985). Interestingly, in adrenalectomized-orchidectomized males rats (and in sham-operated animals), 2 days after surgery, a temporary increase of DHEAS was found in the brain, possibly due to a local neural response to stress (Corpe´chot et al., 1981). Essentially the same results have been obtained with other laboratory animal including monkeys, where a limited study suggested that there is DHEA(S) of both adrenal and cerebral origins in the brain (Robel et al., 1987). Brain steroid concentrations measured in a few human cadavers were in the same 10 − 8 9 1 M range, the values however varied due to the heterogeneity of samples available (Lacroix et al., 1987; Lanthier and Patwardhan, 1986). Globally, it may be observed that in primates, which have sizable concentrations of DHEA and DHEAS in the blood, brain DHEA(S) is more abundant, and PREG(S) less abundant, than in rodents. Also note that DHEA, PREG and their conjugates are found everywhere in the brain, even if there are some differences between certain regions (i.e. relatively more PREG in the olfactory bulb, more DHEA(S) in the hypothalamus in rats). As a whole, the concentrations of several steroids such as DHEA, PREG, their conjugates, and PROG and its 5a-metabolites, expressed in mol/vol. equivalent of brain tissue weight, are relatively high in many instances, and possibly even higher than it appears because of compartmentation, the brain tissues being targets for paracrine/autocrine products.

BIOSYNTHESIS AND METABOLISM OF NEUROSTEROIDS 3b-hydroxy-D5-steroids PREG and DHEA are, in steroidogenic glands, intermediary

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Fig. 1. Metabolism of steroids. Most biosynthetic and metabolic reactions cited in the text are indicated with the corresponding enzymes and some of their pharmacological blockers (marked -).

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Table I. Neurosteroids in the male rat brain PREGS

PREGL

DHEA

DHEAS

DHEAL

PROG

Brain (ng/g) Intact Orx/adx

8.9 ( 92.4) 2.6 ( 9 0.8)

14.2 (9 2.5) 16.9 (9 4.6)

9.4 (9 2.9) 4.9 (9 1.3)

0.24 (9 0.33) 0.14 (9 0.13)

1.70 (90.32) 1.64 (90.43)

0.45 (90.13) 0.29 (90.12)

2.2 (91.1) 3.2 (91.6)

Plasma (ng/ml) Intact Orx/adx

1.2 ( 90.6) 0.3 ( 9 0.1)

2.1 (9 0.9) nd

2.4 (9 0.9) 1.3 (9 0.3)

0.06 (9 0.06) nm

0.20 (90.08) nm

0.18 (90.05) nm

1.9 (90.7) 0.1 ( 90.1)

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PREG

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compounds between cholesterol and active 3-oxo-D4-steroids such as PROG and testosterone. Cholesterol itself can be synthesized in many cells of the nervous system from low molecular weight precursors (for example mevalonate“ cholesterol, PREG and metabolites in cultured glial cells (Hu et al., 1989; Jung-Testas et al., 1989; Jurevics and Morell, 1995). There is also evidence for lipoprotein receptors favoring cholesterol uptake, and thus availability for potential steroidogenesis (for example in glial cells) (Jung-Testas et al., 1992). At the mitochondrial outer membrane level there is a protein, also a benzodiazepine binding entity (Costa et al., 1991; Yanagibachi et al., 1988), which favors the access of cholesterol to the inner membrane-linked side-chain cleavage enzymatic complex (Oftebro et al., 1979) transforming cholesterol (27 carbon atoms) into PREG (21 carbon atoms) (Papadopoulos, 1993). The possibility of a link between pharmacological drug activity and neurosteroid synthesis is of striking interest. After a number of unsuccessful experiments to demonstrate enzymatic conversion of cholesterol to PREG in brain tissues and extracts, immunocytochemical evidence for the presence of the cytochrome P450scc, the specific hydroxylase involved in cholesterol side chain cleavage, was obtained at the level of the white matter throughout the brain (Iwahashi et al., 1990; Le Goascogne et al., 1987, 1989) (Fig. 2), in rat and human. The two associated enzymes, adrenodoxin and adrenodoxin reductase, were correlatively observed. The presence and activity of P450scc in myelinating

Fig. 2. Rat cerebellum white matter stained with anti-P450scc antibody. Upper panel: immuno-peroxidase staining; lower panel: histological staining. The same white matter staining is obtained throughout the brain (Le Goascogne et al., 1987).

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Fig. 3. Newborn rat mixed glial cell cultures. Oligodendrocytes differentiated in vitro, as indicated by 2%,3%-cyclic nucleotide phosphodiesterase activity (CNPase) can synthesize (radioactive) PREG from (radioactive) mevalonate (Jung-Testas et al., 1989).

cells was verified in isolated oligodendrocytes (Fig. 3) and Schwann cells, and biochemically confirmed in oligodendrocyte mitochondria (Hu et al., 1987). Conversely, we have not yet found PREG synthesizing neurons, but some astrocytes are labeled in P450scc immunocytochemical detection experiments. These results have recently been confirmed by the detection of P450scc mRNA in oligodendrocytes (Compagnone et al., 1995; Mellon and Deschepper, 1993). Interestingly, the P450scc gene expression may not involve the steroidogenic factor-1 (Zhang et al., 1995). However, the available data are not quantitatively satisfactory, considering the relatively high level of PREG in the CNS (Warner and Gustafsson, 1995), and the regulatory mechanisms governing P450scc function are unknown (even though some cAMP-induced increase in activity has been observed (Robel et al., 1987), particularly in retina (Guarneri et al., 1994). Although the protein hormones stimulating steroid synthesis in peripheral glands are probably not involved, it remains to investigate the activities of a number of peptidic factors, such as IGF1, NGF, etc… in this respect. Paradoxically, the formation of DHEA and of DHEAS in the CNS has not yet been clearly documented, even though the isolation of these steroids in the brain was at the origin of the neurosteroid concept (Baulieu, 1981), and that the recognized precursor in glandular cells, PREG, was rapidly identified at a higher concentration, as indeed a biosynthetic precursor should be (Corpe´chot et al., 1983). Currently, the possibility that DHEA (or conjugates) derives from cholesterol via unconventional pathways remains open (Prasad et al., 1994). Both PREG and DHEA are found in conjugated forms, sulfate esters and fatty acid esters (‘lipoı¨dal’), whose concentrations are frequently equal or superior to those of the corresponding free steroids (Table I). Preliminary evidence has been obtained for a low sulfotransferase activity (Rajkowski et al., 1997); however it is not excluded that there is formation of steroid sulfate-containing lipidic complexes (‘sulfolipids’) (Prasad et al., 1994). The enzymes corresponding to the widely distributed steroid sulfatase activities of

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the brain have not been cloned. Major conjugation forms of PREG and DHEA in the brain are their fatty acid esters (Jo et al., 1989), designated ‘lipoidal’ derivatives (Hochberg et al., 1976). The acyltransferase responsible for their formation is enriched in the microsomal fraction of brain (Vourc’h et al., 1992) and its activity is highest at the time of myelin formation. While PREG (and PROG) can be largely reduced to give 20a-hydroxy metabolites in glial cells and many neurons, no evidence for 17b-reduction of DHEA to give the weak estrogen D5-androsten-3b-17b-diol has been documented. The 7a-hydroxylation of 3b-hydroxy-D5-steroids can be performed by an enzyme distinct from the classical cholesterol hydroxylase found in the liver (Akwa et al., 1992). The 7a-hydroxy derivatives are of unknown biological significance. As in steroidogenic gland cells and many peripheral tissues (Labrie et al., 1992; Vande Wiele et al., 1965), DHEA and PREG can be oxidized to 3-oxo-D4 steroids (to D4-androstenedione and PROG, respectively) in the nervous system, by the 3b-hydroxysteroid dehydrogenase-isomerase enzyme (3bHSD) (Labrie et al., 1992), which can be inhibited by specific steroidal compounds such as trilostane (Young et al., 1994). The 3bHSD isoforms are present in most parts of the brain and in the PNS, and it is found in glial cells and neurons (Guennoun et al., 1995; Sanne and Krueger, 1995). The metabolism of PREG and DHEA in astroglial cells is regulated by cell density: 3bHSD activity is strongly inhibited at high cell density (Akwa et al., 1993). The formation and metabolism of PROG in the brain has been the subject of a number of studies since 5a-reduced PROG metabolites have attracted the attention of pharmacologists in the context of their effects on GABAAR function. Type-1 isozyme predominates in the brain. The 5a-reduced (dihydrogenated) metabolite of PROG, 5a-DH PROG, is in turn converted to 3a- and 3b-hydroxy-5a-pregnane-20-ones (3a/b, 5aTH PROG). Up to now there has been no demonstration of the synthesis of corticosteroids in the nervous system. For the synthesis of estrogens, the discovery of aromatase (P450arom) (MacLusky et al., 1994; Naftolin et al., 1975) in the brain may be viewed as the first evidence for a steroid metabolism of physiological significance in the nervous system, and therefore the formation of estradiol from testicular testosterone in hypothalamic structures may be considered as that of a neurosteroid in males of several species. However, there has been no systematic study of D4-androstenedione which could be formed from neurosteroidal DHEA or PROG and is also an aromatase substrate. Other enzymatic reactions have been suggested, such as those involving 11b-, 18- and 19-hydroxylases (Compagnone et al., 1995; Gomez-Sanchez et al., 1996; Iwahashi et al., 1993; Miyairi et al., 1988; Ozaki et al., 1991). Whether or not there may be formation of odorous D16-androstene derivatives is unknown (Gower and Ruparalia, 1993). In summary, even if many results are available, the global picture of neurosteroid metabolism is still incomplete and patchwork-like. The formation of PREG and PROG in myelinating glial cells is well established qualitatively, but its quantitative and regulatory aspects remain to be documented. The biosynthetic pathway of the neurosteroid DHEA is poorly understood. Sulfates of 3b-hydroxy-D5 steroids have been duly identified, but the related enzymology is obscure, whereas the metabolism and the significance of fatty acid esters are completely unknown. The 3bHSD and 5a-reductase enzymes are definitively active in the nervous system, probably in many cell types, and are crucial to the formation of neurosteroids in appropriate amounts to be neuroactive (Corpe´chot et al., 1993), such as PROG and 3a,5a-TH PROG. A number of questions remain, including the possible transfers of steroids from one cell type to another with successive further metabolism

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during these passages since the relevant enzymes are differentially located in neurons and glial cells (Melcangi et al., 1994; Pelletier et al., 1994; Robel et al., 1987), and also the important possibility that steroids entering the nervous system from the blood may not follow the same metabolic pathways as the same steroids synthesized in the nervous system. In that case, different effects on nervous function may be discovered.

RECEPTORS OF NEUROSTEROIDS To the diversity of neurosteroids themselves should be added that of the receptor systems. Intracellular receptors The distribution of intracellular receptors in the brain has been described mainly on the basis of binding measurements, autoradiography of tritiated steroids and immunocytochemistry of receptor proteins (McEwen, 1991). Naturally these techniques do not distinguish between receptors for peripheral steroids and neurosteroids, and in any case very little has been done in terms of cloning and sequencing to determine whether receptors in the nervous system are or are not the same as in peripheral target tissues. We have biochemically and immunologically documented the presence of a PROG-R in myelinating glial cells (oligodendrocytes of rats of both sexes and mouse and rat Schwann cells) and cloned it (Fidde`s et al., in preparation). In addition to the very remarkable presence of active PROG-R in glial cells, two notions are of particular interest. The first is the capability of these cells to synthetize PROG from PREG (which itself may derive from cholesterol); therefore the cellular systems include a potential autocrine mechanism which could operate under controlling factors yet unknown. We shall see the functional significance of such a mechanism in Schwann cells in the appropriate section of this paper. The second notion is that the cloning of the ligand binding domain of the glial PROG-R from oligodendrocytes or Schwann cells indicates several amino acid differences compared with both the uterine and the neuronal PROG-R from the same animal. Whether the pharmacology of PROG analogs may differ when interacting with the sexual (uterine, hypothalamic) or the glial (in CNS and PNS) PROG receptors is yet conjectural (unpublished data). Membrane receptors Some steroid metabolites can produce a rapid depression of the CNS activity, and it was reported in 1984 (Harrison and Simmonds, 1984) that alphaxalone (5a-pregnane-3a-ol11,20-one) specifically and potently enhances GABAAR-mediated hyperpolarisation, when the 3b-hydroxy isomer is inactive. A large number of biochemical and electrophysiological experiments have followed, demonstrating that natural (including neurosteroids) and synthetic steroids of very specific structure are in fact potent allosteric modulators of GABAAR function (Majewska et al., 1986) (Fig. 4). Now, besides the anaesthetic and anticonvulsant activities of neurosteroids, hypnotic, anxyolytic and analgesic effects of these compounds have been reported (Ba¨ckstro¨m, 1995; Gee et al., 1995; Lambert et al., 1996; Majewska, 1992; Purdy et al., 1991; Schumacher et al., 1993). Neurosteroids may also play an unexpected endocrine regulatory role via the GABAAR (Brann et al., 1995; El-Etr et al., 1995; Genazzani et al., 1996).

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Fig. 4. A schematic cartoon of the ligands stimulating ( ) or inhibiting (¡) GABAAR function (MacDonald and Olsen, 1994).

3a,5a-TH PROG, its 5b-isomer, but not the corresponding 3b-hydroxy steroid, do enhance GABA evoked currents at concentrations as low as 1 nM. There is no absolute GABAAR subunit specificity yet demonstrated for neurosteroids as there is for benzodiazepine binding. The polymorphism of GABAAR in terms of subunit composition (MacDonald and Olsen, 1994) across the different cells of the nervous system, in neurons and glial cells, deserves more study in order to discover steroid derivatives with specific functions. Another type of complexity comes from data obtained when considering the interaction of PREGS with GABAAR. At very low concentrations, in the nanomolar range, the steroid is a weak enhancer of GABA evoked currents, but at a micromolar concentration it produces a non competitive voltage-independent inhibition (Majewska et al., 1988) (due to reduced frequency of channel opening (Mienville and Vicini, 1989)).

Fig. 5. PREGS inhibits GABAAR function in vivo (Majewska et al., 1989).

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Fig. 6. PREGS stimulates NMDA-R function (Wu et al., 1991).

These effects may be observed in vivo (Majewska et al., 1989) (Fig. 5). DHEAS also is an allosteric inhibitor of the GABAA-R (Majewska et al., 1990). A global understanding of the potential effects of neurosteroids on neurotransmission should also take into account other modulatory activities displayed by the steroids. PREGS appears to allosterically potentiate the NMDA receptor (Wu et al., 1991) (Fig. 6), and this effect may functionally reinforce the antagonistic effect of the same steroid on GABAAR and on the glycine receptor. PREGS also inhibits non NMDA glutamate receptors. Other modulatory activities of neurosteroids have been described on glycine-activated chloride channels (Prince and Simmonds, 1992), on neural nicotinic acetylcholine receptors reconstituted in Xenopus lae6is oocytes (Valera et al., 1992), and on voltage-activated calcium channels (ffrench-Mullen et al., 1994). Sigma receptors, as pharmacologically defined by their effect on NMDA R activity, have been studied in rat hippocampal preparations: here DHEAS acts as a sigma receptor agonist, differently from PREGS which appears as a sigma inverse agonist (Fig. 7), and PROG which behaves as a s antagonist (Monnet et al., 1995).

Fig. 7. Neurosteroids modulate NMDA-R function via sR.

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Fig. 8. PREG is selectively decreased in the olfactory bulb of a male rat exposed to the ‘odor’ (pheromone?) of a female rat (Corpe´chot et al., 1985).

Are there other specific membrane receptors, of which the ligands are primarily neurosteroids, in contrast with the preceding examples indicating an allosteric function upon neurotransmitter receptors and ion channels? Rapid effects of steroids may be explained by such novel receptors (Schumacher et al., 1990; Smith et al., 1987). A G-protein coupled corticosteroid receptor has been identified in synaptic membranes from an amphibian brain (Orchinik et al., 1991). Studies have indicated specific binding of PROG conjugated with a macromolecule (albumin in general) to different cellular or membrane preparations, but none has been able to determine its biological relevance (Tischkau and Ramirez, 1993). We also found selective binding of steroid sulfates to purified neural membranes of yet unknown biological significance (Robel and Baulieu, 1994).

PHYSIOLOGICAL AND PATHOLOGICAL ASPECTS Beha6iour We observed an increase of brain DHEAS related to surgical (adrenalectomy and gonadectomy) stress conditions in the rats (Corpe´chot et al., 1981). We also observed that the exposure of male rats to females (Fig. 8) leads to a decrease of PREG in the rat olfactory bulb, an effect apparently due to a pheromonal stimulus, ovarian-dependent in the females, and testosterone-dependent in males (orchiectomy suppresses the response and testosterone reestablishes it) (Corpe´chot et al., 1985). A particular model of aggressiveness in castrated male mice (Fig. 9), inhibited by testosterone or estrogen administration, has also been studied (Haug et al., 1989; Young et al., 1991). The stimulus is the introduction of a lactating female in a cage containing three resident orchiectomized males. The administration of DHEA (280 nmol for 15 days), decreases the males aggressiveness. This does not seem due to a transformation of DHEA

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into brain testosterone (which has been measured), and indeed a DHEA derivative (3b-methyl-D5-androsten-17-one), which is not converted to androgens or estrogens and is devoid of any hormonal activity, is at least as active as DHEA itself. Interestingly we found a progressive and significant decrease of PREGS in the brain, which may be related to the decrease of aggressiveness (experiments have excluded that an increase of 3a-5a-TH PROG, activating the calming GABAAR, was responsible). Other behavioral changes have been found correlated to changes of neurosteroid concentrations in the brain. In particular, in the cases of increased production or administration of PROG or deoxycorticosterone, the elevation in the brain of the related 3a-hydroxy-5a-reduced tetrahydro-metabolites (3a-5a-TH PROG and 3a,5a-tetrahydrodeoxycorticosterone), both agonists for the GABAAR, which may therefore be responsible for corresponding behaviors in pregnancy or stress, respectively (Majewska, 1992; Paul and Purdy, 1992). Cogniti6e performance The study of cognitive performance in deficient aging rats has been particularly rewarding (Valle´e et al., 1997) (Fig. 10). We found that PREGS is significantly lower in the

Fig. 9. PREGS is selectively decreased in brain when the aggressiveness of castrated male mice, towards exposed to a female intruder, is inhibited by DHEA administration (Young et al., 1994).

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Fig. 10. Hippocampal PREGS is lower in old rats than in young rats, but unevenly (A). Memory tasks classify old rats in impaired and unimpaired animals (B). Hippocampal PREGS and memory task impairment (water maze) are inversely correlated (C). Administration of PREGS temporarily improves memory performance in impaired rats (D).

hippocampus of aged (24 month old rats) than in young (male) animals. Interestingly, the individual concentration (ng/g) of PREGS in the hippocampus of aged animals was widely distributed, between 2 and  28 ng/g of tissue. The animals had previously been classified according to their performance in two tasks for spatial memory, the Morris water maze and the two-trial test in a Y maze (Mayo et al., 1993): low levels of PREGS

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Fig. 10. (Continued)

in hippocampus were correlated with poor performance in both tasks. When aged rats which had been classified as mempry-impaired received a single does (by intraperitoneal injection) of PREGS, their performance was significantly improved, albeit transiently. Both the physiological approach (measurement of endogenous neurosteroid levels) and the pharmacological approach (effect of administered PREGS) suggest strongly that hippocampal PREGS is involved in the maintenance of cognitive performances. These observations are consistent with the results of systemic or intracerebral administration of PREG or derivatives in rodents, enhancing their natural memory performances or antagonizing pharmacologically induced amnesia (Flood et al., 1992; Mathis et al., 1994). In most experiments however, the procedure involves alteration of the motivational or emotional states of the animals, and the direct relationship between the administered dose and endogenous steroid concentration has yet to be established. We have thus described, for the first time, changes of a neurosteroid with age, individual differences and the correlation of these differences with behavioral performance, plus the improvement of performance in impaired PREGS-deficient aged animals by the steroid administration.

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Fig. 11. PREG in male rat sciatic nerve. No effect of castration and adrenalectomy (Koenig et al., 1995).

These results are determinant for attributing a physiological significance to a neurosteroid. However, the molecular aspects of the involvement of PREGS in memory, i.e. the steroids metabolism as well as receptors and enzymes involved, remain to be worked out. Trophic action. Myelination In a completely different field, namely myelin repair in a wounded peripheral nerve, there is also a clearcut demonstration of the physiological function of a neurosteroid. We found PREG in the sciatic nerve of human cadavers at a mean concentration ] 100-fold the plasma level of the steroid (Morfin et al., 1992), suggesting a possible biosynthesis, that we guessed to be in Schwann cells by analogy with what has previously been obtained in oligodendrocytes. The experimental simplicity of working with the PNS led to studying the regeneration of cryolesioned sciatic nerve in mice. We measured PREG and PROG in the sciatic nerve of normal animals and after adrenalectomy and gonadectomy (Fig. 11). The levels of the two steroids are much higher in the nerve than in the plasma, while the corticosterone concentration is much lower in nerve than in plasma. After surgical endocrine ablation, PREG and PROG remained high in nerve and corticosterone decreased in blood. After cryolesion (Koenig et al., 1995) (Fig. 12(A)), axons and their accompanying myelin sheaths degenerate quickly in the frozen zone and the distal segments (Wallerian degeneration). However, the intact basal lamina tubes provide an appropriate environment for regeneration. Schwann cells start to proliferate and myelinate the regenerating fibers after 1 week, and 2 weeks after surgery, myelin sheaths have reached about one third of their final width. In the damaged portion of the nerve, PREG and PROG levels remain high, and even increase 15 days after lesion (if expressed in pg/cm). The role of PROG in myelin repair, assessed after 2 weeks, was indicated by the decrease of thickness (number of lamellae) of myelin sheaths when trilostane, an inhibitor

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of 3b-HSD involved in the PREG to PROG transformation, was applied to the lesioned nerve (Fig. 12(B)), or when, alternatively, RU486 was locally delivered in order to competitively antagonize PROG action at the receptor level (Fig. 12(C)) (as indicated before, we had detected a PROG-R immunologically and by binding studies in Schwann cells). The inhibitory action of trilostane (Fig. 12(B)) could not be attributed to toxicity

Fig. 12. Cryolesion of sciatic nerve. Three steps (A) Remyelination: effect of the blockade of 3b-HSD by trilostane (B). Antagonism of PROG at PROG-R by RU486 (C) (Koenig et al., 1995).

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Fig. 12. (Continued)

since its effect was reversed by the simultaneous administration of PROG. We could even enhance remyelinisation with a high dose of either PREG or PROG. In this in vivo system, the structure of the myelin sheaths formed in response to neurosteroids, as determined by electron microscopy, was morphologically normal. Such an effect was also apparent in cultures of rat dorsal root ganglia (DRG). After 4 weeks in culture, neurite elongation and Schwann cell proliferation had ceased and, in the appropriate medium, the presence of a physiological concentration of PROG (20 nM) for 2 weeks did not further increase the area occupied by the neurite network, the density of neurites, or the number of Schwann cells, but it did increase the number of myelin segments and the total length of myelinated axons  6-fold (Fig. 13). Mechanistically, several hypotheses may be raised: PROG produced by Schwann cells may act on adjacent neurons and activate the expression of neuronal signalling molecules required for myelination. Alternatively, PROG may function as an autocrine trophic factor and directly enhance the formation of new myelin sheaths. We are currently studying the effect of PROG on the expression of myelin specific proteins. We also analyze the reciprocal influences of neurons and Schwann cells on each other, in terms of Schwann cell proliferation and protein synthesis, and metabolism of PREG and PROG in both cell types (Fig. 14). We would just like to mention that the rather high concentrations of PROG in intact adult nerves suggest a role for this neurosteroid in the slow but continuous renewal of peripheral myelin. It is quite interesting that PROG, a classical sex steroid, is also an active neurosteroid very probably synthesized and active independently of any sexual context. If similar results are obtained with CNS elements of human origin, we may soon have to work on the possible therapeutic effect of PROG analogs, for treating or preventing certain demyelinating pathological conditions.

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CONCLUSIONS Neurosteroids are synthesized in the central and peripheral nervous system, particularly in myelinating glial cells, but also in astrocytes and many neurons, and act in the nervous system. Synthetic pathways may start from cholesterol or from steroidal precursor(s) imported from peripheral sources. Measured concentrations of neurosteroids are consistent with the affinities of receptor systems with which they interact in the nervous system. Both intracellular and membrane receptors responding to neurosteroids can be distin-

Fig. 13. PROG in vitro stimulates myelination of neurites (from mouse dorsal root ganglia in culture). No progesterone (A). Progesterone 20 nM (B) (Koenig et al., 1995).

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Fig. 14. Schwann cells-neurons interaction in culture. Neurons stimulate the synthesis of PROG from PREG by a diffusible substance (A), while the formation of 3a,5a-TH PROG necessitates axonal contact (B).

guished (Fig. 15). For the former, receptors are identical or similar to those of steroid hormones found in peripheral target organs; a PROG-R in particular has been found in oligodendrocytes and Schwann cells, evoking the possibility of an autocrine system for PROG action. For the latter, neurosteroids in the brain are in fact allosteric modulators of neurotransmitter receptors, and their levels are compatible with their playing a physiological neuromodulatory role; this is the case with the GABAAR, but also the NMDA-R, sigma1-R and others. Besides numerous of experiments of a pharmacological nature consistent with well defined properties of several neurosteroids, there are now results which demonstrate a contribution of steroids formed endogenously and accumulated in the nervous system independently, at least in part, of any contribution from the steroidogenic glands. PROG synthesized in Schwann cells has ‘trophic’ activity; it contributes to the synthesis of myelin in regenerating sciatic nerve in rats and mice, and the effect can be demonstrated in vivo and in vitro. PREGS has a behavioral effect in aged (2 year old) rats: the concentration of PREG-S in the hippocampus (and not in other parts

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Fig. 14. (Continued)

of the brain) is inversely correlated with the quantified impairment of an individuals accomplishment of each of two memory tasks in a Y maze and a water maze, and we obtain temporary improvement after PREG-S administration. Therefore, it is important to study the neuromodulatory role of neurosteroids in situations such as the estrous cycle, pregnancy, menopause, stress and their influence on sexual behavior, memory and the developmental and aging processes. Available data suggest that the neurosteroid concept may also be applicable to humans, and the robust and specific activities of neurosteroids may become useful for enlarging therapeutic approaches to functional and trophic alterations of the nervous system. Acknowledgements: I would like to thank researchers and students of my laboratory, and colleagues from many others institutions who helped us to collect the data reported in this presentation. When I say ‘we’ in the text, it means first of all P. Robel and his wise and persistent contributions, and for many years C. Corpe´chot, I. Jung-Testas, B. Eychenne, M. El-Etr, C. Le Goascogne and more recently Y. Akwa, K. Rajkowski, M. Schumacher. The work of my laboratory has been supported mainly by INSERM. I gratefully acknowledge the help of the Centre National pour la Recherche Scientifique (CNRS), the College de France, Roussel-Uclaf,

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Fig. 15. In the nervous system, steroidal ‘precursors’, metabolites of ‘hormones’, and ‘hormones’ themselves exert activity through either membrane or intracellular receptors, or both.

l’Association pour la Recherche de la Scle´rose en Plaque (ARSEP), l’Association Franc¸aise contre les Myopathies (AFM), the Mathers Foundation, the Myelin Project, and P. Schlumberger. I also thank F. Boussac, C. Legris and J.C. Lambert for preparing the manuscript.

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