Progress in NeurobiologyVol. 38, pp. 379 to 395, 1992 Printed in Great Britain.

0301-0082/92/$15.00 1992 Pergamon Press plc

NEUROSTEROIDS: E N D O G E N O U S BIMODAL MODULATORS OF THE GABAA RECEPTOR. MECHANISM OF ACTION A N D PHYSIOLOGICAL SIGNIFICANCE MARIA DOROTA MAJEWSKA Laboratory of Neuropharmacology, Addiction Research Center, NIDA, Baltimore, MD, U.S.A.

(Received 10 July 1991) CONTENTS Abbreviations 1. Introduction 2. Biosynthesis of neurosteroids 3. Neurosteroids as regulators of the GABA^ receptors 3.1. GABA-agonistic neurosteroids 3.1.1. Biochemical and electrophysiological evidence 3.1.2. Synaptic actions 3.1.3. Steroids versus barbiturates 3.2. GABA antagonistic neurosteroids 3.2.1. Biochemical and electrophysiological evidence 3.3. Structural relationships between GABA-antagnnistic and GABA-agnnistic steroids 3.4. Steroid recognition sites at the GABA A receptor 3.4.1. Neurosteroid binding: pharmacology 3.4.2. Neurosteroid binding sites: proteins or lipids? 3.4.3. Evidence from electrophysiology and receptor cloning 3.4.4. Steroid regulatory sites at the GABA A receptor: hypothetical model 4. Behavioral effects of GABA-modulatory steroids 4.1. GABA-agonistic steroids 4.2. GABA-antagonistic steroids 5. Steroids/GABAA receptor interactions: Psycho-physio-pathological role 5.1. Stress 5.2. Diurnal cycles 5.3. Depression and anxiety 5.4. Aggression 5.5. Personality traits 5.6. Sexual functions, pregnancy, "post-partum blues" 5.7. Seizures 5.8. Feeding and blood pressure regulation 5.9. Cognitive functions 5.10. Defects in steroidogenesis 6. Summary Acknowledgements References Addendum

379 379 38O 381 381 381 381 382 382 382 382 383 383 384 384 385 385 385 386 386 387 387 387 388 388 388 389 389 389 390 390 39O 391 394

ABBREVIATIONS CNS GABA DHEAS DHEA P

Central nervous system 3'-aminobutyric acid Dehydroepiandrosterone sulfate Dehydroepiandrosterone Pregnenolone

"It should not be forgotten that science is not the summa of life, that it is actually only one of the psychological attitudes, only one form of human thought." (Carl Gustav Jung)

* New address: NDIA/MDD, Rmlla-5600, Fishers Lane, RockviUe, MD 20857, U.S.A.

PS THP THDOC

Pregnenolone sulfate tetrahydroprogesterone (5~t-pregnane-3~tol-20-one) tetrahydrodeoxycorticosterone (5~-pregnane3,,,2 l-diol-20-one)

1. I N T R O D U C T I O N Steroids are vital for cell life. In eukaryotic organisms steroid hormones emerged early in evolution as primitive growth regulators, and diversified later to sex steroids, gluco- and mineralocorticoids with remarkable preservation of structure-activity relationships (Rousseau and Baxter, 1979). In mammals all steroid hormones derive from cholesterol and they 379

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are synthesized in primary steroidogenic organs such as adrenal glands (mineraio- and glucocorticoids), gonads and placenta (sex hormones), before being secreted into the blood stream. High lipophilicity of steroids ensures their easy penetration of biological membranes, enabling access to all cells and organs, including the central nervous system (CNS). Steroid actions in the CNS produce diverse, both rapid and delayed effects (McEwen et al., 1979; McEwen, 1982; Riker et al., 1982; Parsons and Pfaff, 1985). A plethora of data has accumulated over the last decade which indicates that steroids are also synthesized in the CNS. The steroids of central origin have been termed "neurosteroids" (Baulieu et al., 1987). When they were first discovered, their role in the CNS was obscure, but now it has become evident that the neurosteroids constitute a group of vital multimodal neuromodulators. With these premises in mind, the current definition of neurosteroids includes both the aspect of central origin of these steroids and their functional neuromodulatory role. This article reviews the experimental evidence of biosynthesis of neurosteroids in the brain. It also describes the neuromodulatory role of neurosteroids in the CNS, and particularly, the bimodal regulation of the GABA A receptors, documented by biochemical, electrophysiological and behavioral observations. Finally, the psycho-physio-pathological consequences of steroid interactions with the central GABA A receptor will be discussed. An interested reader is referred to my earlier commentary articles on this topic (Majewska, 1987a, 1990). 2. BIOSYNTHESIS OF N E U R O S T E R O I D S

The mammalian brain contains substantial amounts of steroid precursor, cholesterol, as well as its sulfate and lipid derivatives (Iwamori et al., 1976). The brain is also "equipped" with enzymes that are necessary for steroid metabolism. Although it was known for a long time that the brain can metabolize the steroids "imported" from periphery, only during the last decade has it become clear that the brain can also synthesize de novo certain steroids, which have been termed "neurosteroids". The presence of a metabolically active pool of steroids, independent of peripheral steroidogenic glands, was demonstrated in mammalian brain tissues from rodents through primates (Baulieu et al., 1987). The neurosteroids that were originally discovered and characterized were pregnenolone (P; 21 carbon) and dehydroepiandrosterone (DHEA; 19 carbon), their sulfate derivatives (pregnenolone sulfate, PS; and dehydroepiandrosterone sulfate, DHEAS), and their fatty acid esters (Corpechot et al., 198t, 1983; Baulieu et al., 1987). These steroids were found in the brain at concentrations much greater than those in plasma, suggesting that they play a functional role in the CNS. The neurosteroids, which have 3fl-hydroxy-A5 structures, derive from cholesterol after side-chain cleavage. The mitochondrial enzyme involved in sidechain cleavage of cholesterol (cytochrome P-450~) was found throughout the rat brain, mostly in the white matter (Le Goascogne et al., 1987). Production of pregnenolone (P) from cholesterol or mevalonolac-

tone was found in glioma C6 cell line and in oligodendrocytes, but apparently not in neurons (Hu et al., 1987; Jung-Testas et aL, 1989) thereby suggesting primarily glial origin of neurosteroids. Cholesterol sulfate also undergoes side-chain cleavage yielding pregnenoione sulfate, and steroid sulfates can be hydrolyzed to primary steroids by sulfatases that are present in the CNS (Iwamori et al., 1976b). In adrenal glands, gonads and placenta, P can be cleaved to DHEA by the specific enzyme complex 17~-hydroxylase-17-20-desmolase, which contains cytochrome P45017a (Lieberman et al., 1984). However, attempts to localize cytochrome P450,7~ in the brain by using immunohistochemical methods were unsuccessful to date (Baulieu and Robel, 1990), thereby leading the authors to consider the existence of an alternative metabolic path of conversion of P to DHEA in the CNS. The brain also contains other enzymes which are required for further metabolism of P and DHEA. P can be converted in the brain to progesterone by 3fl-hydroxy-steroid oxidoreductase, 5-ene-isomerase (Weidenfield et al., 1980). This process was recently demonstrated in the oligodendroglia (Jung-Testas et al., 1989). Brain tissues also embody steroid 5a-reductase and 3~-oxidoreductase (Rommerts et al., 1971; Roselli and Snipes, 1984; Celotti et al., 1987; Krause and Karavolas, 1980), mainly in the glial compartment (Canick et al., 1986; Krieger and Scott, 1989). These enzymes reduce progesterone to 5~-pregnane-3a-ol-20-one (tetrahydroprogesterone; THP), whose de novo biosynthesis was recently demonstrated in primary cultures of oligodendrocytes (Jung-Testas et al., 1989b). DHEA can be metabolized in the CNS via the same metabolic pathways as pregnenolone, being converted to androstenedione (4-androsten-3,17-dione), and subsequently it can be reduced to androsterone (5~ -androstan-3~-ol- 17-one). Originally only pregnenotone, dehydroepiandrosterone, and their sulfo- and lipid derivatives, were regarded as neurosteroids, but now it seems appropriate to include into this term all the steroids formed in the CNS. Perhaps, the definition of neurosteroids should refer not only to steroids synthesized in the CNS de novo, but should be extended also to the metabolites of progesterone, deoxycorticosterone or testosterone, which are formed in the brain from peripheral hormones. As the glia seems to be the primary site of biosynthesis of neurosteroids, it can be regarded as a neuroparacrine gland which produces steroidal neuromodulators. The amounts of neurosteroids, PS and DHEAS, measured in rodent brain are in the range of 2-16 ng per gram of fresh tissue (Corpechot et al., 1981, 1983). However, micromolar K m values for steroid sulfatases and compartmentalization of neurosteroids suggest that micromolar concentrations of PS and DHEAS may be present in certain brain compartments. Reduced metabolites of progesterone were found in the brain at nanomolar to micromolar concentrations, being many times greater from plasma levels (Backstrom et al., 1990). Brain concentrations of neurosteroids undergo dramatic physiological and cyclical variations, which will be discussed later.

MODULATORS OF THE G A B A A RECEPTOR

Supplementing animal studies, high concentrations of neurosteroids were also found in post mortem human brains of men and women of age > 60 years (Lanthier and Patwardhan, 1986; Lacroix et aL, 1987). The neurosteroids, including progesterone, pregnenolone, and dehydroepiandrosterone, were present in all regions of human brain at concentrations many times higher from those detected in plasma (the mean ratio of brain to plasma levels of pregnenoione was 74, and it was about 7 for dehydroepiandrosterone). There was a definite trend for higher concentrations of DHEA and DHEAS in brains of women than men, although the physiological significance of this intriguing finding is not known. Taken together, both the animal and human data strongly suggest local biosynthesis of neurosteroids in the brain. 3. NEUROSTEROIDS AS REGULATORS OF THE GABAA RECEPTORS 3.1. GABA-AGONISTICNEUROSTEROIDS 3.1.1. Biochemical and electrophysiological evidence

GABA, a principal inhibitory neurotransmitter in the CNS, activates two types of receptors, the ionotropic GABA A receptor and metabolotropic (G-protein coupled) GABA a receptor. The ubiquitous GABA^ receptor is a protein tetra- or pentamer (Schofield et aL, 1987), whose activation by agonists opens the associated chloride channel, leading to increased chloride transport and resulting usually in hyperpolarization of neuronal membrane. Recent findings in molecular biology revealed the heterogeneity of GABA A receptors, which come at different combinations of polypeptide subunits (~t, t, 7, 6, ~) Olsen and Tobin, 1990; Vicini, 1991). Activity of the GABAA receptor can be modified by several psychotropic drugs, such as benzodiazepines, barbiturates, or convulsants, which act at different domains of the receptor. Benzodiazepines and barbiturates potentiate function of GABA A receptors, while convulsants, such as picrotoxin or t-butylbicyclophosphorothionate (TBPS), inhibit receptor activity. Recently it became evident that also several steroids, including the neurosteroids, directly regulate the activity of the GABAA receptor/chloride ionophore complex in a bimodal fashion. The genesis of this finding stemmed from two independent experimental avenues. One group discovered an interaction between steroids and GABA^ receptors while investigating the electrophysiological effects of steroidal anesthetic alphaxalone (Harrison and Simmonds, 1984), and I deduced and described the existence of this interaction (Majewska et al., 1985) based on my observation that cholesterol--which was used originally as a modifier of membrane fluidity--altered GABA binding to the GABA^ receptor (M. D. Majewska, unpublished observation). The endogenous and synthetic steroids, which contain reduced A ring at C-5 in the a or ~ position, hydroxyl at the 3a-position and electronegative atom (usually oxygen) at C-17 or C-20, behave as allosteric agonists of the GABAA receptor (Harrison and JPN 38/4--D

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Simmonds, 1984; Majewska et al., 1986; Harrison et al., 1987; Peters et al., 1988; Turner et al., 1989). The most potent (active at nanomolar concentrations) naturally occurring steroids with allosteric GABAagonistic features are tetrahydroprogesterone (5~pregnane-3~t-ol-20-one, THP), tetrahydrodeoxycorticosterone (5~t-pregnane-3~t,21-diol-20-one, THDOC) (Majewska et al., 1986; Harrison et al., 1987; Lambert et al., 1990), and androsterone (5~t-androstan-3ct-ol-17-one) (Peters et al., 1988; Turner et al., 1989). The modulatory actions of GABA-agonistic steroids resemble the effects of anesthetic barbiturates, which are manifested by their following actions: (I) enhancement of GABA (or its agonist) and benzodiazepine binding to brain membranes (Majewska et al., 1986; Majewska, 1988; Harrison et al., 1987; Peters et al., 1988; Turner et al., 1989); (II) inhibition of binding of the radiolabeled convulsant t-butylbicyclophosphorothionate (TBPS) to GABA^ receptoroperated chloride channel in a noncompetitive manner (Majewska et al., 1986; Gee et al., 1988; Turner et al., 1989); (III) enhancement of GABAinduced chloride transport in synaptoneurosomes (Majewska et al., 1986; Turner et al., 1989; Im et al., 1990); and (IV) potentiation of GABA a receptor mediated current in neurons (Majewska et al., 1986; Harrison et al., 1987; Peters et al., 1988). GABA-enhancing effects can be observed at nanomolar concentrations of active steroids (threshold concentrations for enhancement are 10-30 riM), but at slightly higher concentrations these steorids also directly open GABA A receptor-operated chloride channel in neurons (Majewska et al., 1986; Harrison et al., 1987; Lambert et al., 1990). The GABA^ receptor active steroids prolong the electrophysiological response to GABA due to increase burst duration of the channel currents elicited by GABA (Harrison et al., 1987; Barker et al., 1986). 3.1.2. Synaptic actions The neurosteroids which potentiate function of GABAA receptor were also shown to prolong GABA mediated inhibitory postsynaptic potentials (IPSP) in hippocampal neurons (Harrison et al., 1987), indicating that neurosteroids are modulators of synaptic events. The neuromodulatory actions of neurosteroids are illustrated even more dramatically by experiments showing that the GABA-agonistic steroid, alphaxalone, depressed the depolarizing responses to iontophoretically applied glutamate, and blocked the glutamate-induced action potentials under current clamp conditions (Lambert et al., 1990). These effects apparently were not mediated via direct steroid actions at the glutamate receptors, because alphaxalone did not alter currents induced by ligands specific for these receptors, such as NMDA, quisqualate or kainate, measured under the voltage clamp condition. Rather, the inhibition of neuronal depolarization by alphaxalone resulted from activation by this steroid of the GABA^ receptors. Hence, the electrophysiological effects and the previously described biochemical data decisively indicate that the neurosteroids act on the GABA-receptor to

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modulate synaptic events, and as such may play a vital role in neuronal plasticity. 3.1.3. Steroids versus barbiturates

Prima facie, the biochemical, electrophysiological and behavioral actions of GABA-agonistic steroids appeared similar to those of barbiturates, but more meticulous tests revealed that sites of action of these two groups of modulators may not be the same. Specifically, the potentiating effects of barbiturates and steroids on the binding of [3H]muscimol and [3H]flunitrazepam in synaptosomes, and on stimulation of [36C1-] transport in synaptoneurosomes, were shown to be additive or synergistic (Peters et al., 1988; Turner et al., 1989; Im et al., 1990; Majewska, unpublished observations). Similarly, additivity of steroid and barbiturate actions on CI currents in neurons has been observed (Peters et al., 1988). Dissimilarity of sites of action of GABA-agonistic steroids and barbiturates was also inferred from the kinetics of their effects on dissociation of TBPS binding (Gee et al., 1988). Taken together, the biochemical and etectrophysiological data imply that GABA-agonistic steroids do not share common site of action with anesthetic barbiturates, although their modes of action are remarkably similar. 3.2. GABA-ANTAGONISTIC NEUROSTEROIDS

3.2.1. Biochemical and electrophysiological evidence In contrast to THP, THDOC, or androsterone, which act as allosteric agonists of the GABA A receptor, some neurosteroids behave as noncompetitive antagonists of this receptor (Majewska and Schwartz, 1987; Majewska et al., 1988, 1990; Mienville and Vicini, 1989). To the latter category belong pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). First, I observed that PS bimodally alters binding of the G A B A agonist, [3H]muscimol, to the GABA g receptor in synaptosomal membranes; increasing binding at nanomolar concentrations and decreasing it at micromolar concentrations (Majewska et al., 1985). Subsequent studies revealed mixed GABAagonistic/antagonistic features of PS, because, while this steroid slightly potentiates benzodiazepine binding, it also inhibits barbiturate-induced enhancement of benzodiazepine binding (Majewska and Schwartz, 1987). GABA-antagonistic properties of PS were also deduced from the fact that PS at low micromolar concentrations, competitively inhibits binding of the convulsant [35S]TBPS to the GABAA receptor operated chloride ionophore. Subsequent functional assays proved that PS acts as antagonist of the GABAA receptor, as it blocks, in a dose-dependent manner (IC50 = 20---60/.tM), GABA-induced chloride transport or current in synaptoneurosomes and neurons, respectively (Majewska and Schwartz, 1987; Majewska et al., 1988). More recently we found that the neurosteroid, DHEAS, also inhibits GABA-induced currents in neurons in a noncompetitive manner (IC50 about 10/~M) (Majewska et al., 1990; Demirgoren et al., 1991). Although both PS and DHEAS inhibit

GABA-induced currents, there are differences between these steroids in their modes, and presumably sites, of action. For example, while PS is a potent inhibitor of binding of the chloride channel ligands, [35S]TBPS and [3H]TBOB, DHEA is devoid of this capability (Majewska and Schwartz, 1987; Majewska et al., 1990). Moreover, DHEAS, unlike PS, does not enhance [3H]benzodiazepine binding; but rather reduces it at micromolar concep.trations (Demirgoren et al., 1991). Finally, there is a difference in actions of the desulfated forms of PS and DHEAS; pregnenolone is inactive as modulator of the GABA A receptor (Harrison et al., 1987; Turner et al., 1989), whereas dehydroepiandrosterone (DHEA) inhibits GABA-induced currents, albeit 3-4 times less potently than DHEAS (Demirgoren et al., 1991). The GABA-antagonistic features of PS, DHEAS and dehydroepiandrosterone observed in vitro are consistent with the in vivo observations of their excitatory actions on neurons (Carette and Poulain, 1984), and with their convulsant actions (Fidgor et al., 1957; Heuser and Eidelberg, 1961). Glucocorticoids also seem to interact with the GABA A receptor, but their pattern of interaction appears more complex. In synaptosomal fractions prepared from various brain regions of adrenalectomized rats, glucocorticoids altered G A B A binding to GABAA receptors in a biphasic mode, potentiating it at nanomolar concentrations and reducing it at micromolar ones, thereby resembling the actions of pregneno;one sulfate (Majewska et al., 1985). When glucocorticoids were examined for their interactions with the convulsant recognition site at the GABA A receptor, they also showed a biphasic mode of interaction. Potentiation of [35S]TBPS binding--which was observed at nanomolar concentrations of glucocorticoids--resembled the effect of the GABAA receptor antagonist, bicuculline, suggesting that glucocorticoids may have some GABA-antagonistic features (Majewska, 1987). In functional assays, glucocorticoids altered GABAA receptor mediated contractile responses in guinea pig ileum in a biphasic manner (Ong et al., 1987), although the electrophysiological recordings in neurons of the effects of glucocorticoids were not conclusive. Therefore, further studies are needed to establish whether, and how, glucocorticoids modulate function of the GABA A receptor. 3.3. STRUCTURALRELATIONSHIPS BETWEEN G A B A ANTAGONISTIC AND GABA-AGONISTIC STEROIDS

There exists a close metabolic link between GABAantagonistic and -agonistic steroids. The structural relationships between these steroids are shown in Fig. 1. The GABA-antagonistic steroids, PS and DHEAS, which are formed in the CNS from cholesterol sulfate can be desulfated by steroid sulfatases (Iwamori et al., 1976), and subsequently metabolized by brain enzymes to the GABA-agonistic steroids, THP (5~t-pregnane-3~t-ol-20-one) and cis-androsterone (5~-androstan-3ct-ol-17-one). A similar type of metabolic relationship exists between excitatory and inhibitory amino acids, where glutamate is a precursor of GABA. Such metabolic links may be a part of the homeostatic mechanism of control of neural

MODULATORSOF THE G A B A

383

A RECEPTOR

GABA ANTAGONISTS CH s

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C-----O . . . . oo . . . .

DHEAS CH 3

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I

C-----O

o

ANDflOSTENDIONE

PROGESTERONE

GABA AGONISTS

1

CH 3

I

C=O

H

0

H THP

ANDROSTERONE

FIG. 1. Structural and metabolic relationships between the GABA-antagonistic steroids (pregnenolone sulfate, PS and dehydroepiandrosterone sulfate, DHEAS) and the GABA-agonistic steroids (5~-pregnane3~t-ol-20-one; tetrahydroprogesterone; THP) and (5~t-androstan-3ct-ol-17-one; androsterone). PS and DHEAS can be converted to progesterone and androstenedione, and subsequently reduced to THP and androsterone, respectively.

activity through precise regulation of synthesis of counteracting neuromodulators, which is highly adaptive to internal and external milieus. 3.4. STEROID RECOGNITION SITES AT THE GABA A RECEPTOR

3.4.1. Neurosteroid binding: pharmacology

The results of binding experiments of [3H]PS and [3H]DHEAS rendered insight into the pharmacological and biochemical nature of sites of steroid interaction with the GABAA receptor. Both PS and DHEAS are water soluble, and thus are good ligands for binding experiments. These two steroids appear to bind specifically to at least two populations of sites in crude synaptosomal membranes from rat brain (Majewska et al., 1990a,b; Demirgoren et al., 1991). The population of low affinity and very high density binding sites for both steroids (Kd in high #M range; Bm~x > 10 nmol/mg protein), most likely represents the loci of steroid incorporation into membrane lipids. In contrast, the higher and intermediate affinity binding sites for both steroids seem to be

associated with the GABAA receptors. K~ of high affinity PS binding sites is in the range 300-500 nM, and Kd of intermediate affinity sites is about 20 # u (Majewska et al., 1990a). For DHEAS, the apparent Kd of high affinity sites is about 3 #M (Majewska et al., 1990b). Although PS and DHEAS inhibit each other binding, their specific sites of binding seem distinct. High affinity binding sites for [3H]PS and [3H]DHEAS have different densities. While the Bm~x for high affinity [3H]PS binding (about 5 pmol/mg protein) is similar to the density of GABA^ receptors (Enna and Karbon, 1986; Majewska et al., 1985), the Bm~ for [3H]DHEAS binding (about 60pmol/mg protein) is much higher (Majewska et al., 1990b). These facts suggest that some of the high affinity DHEAS recognition sites may not be associated with GABAA receptors, or that several molecules of DHEAS may bind to each receptor. An alternative explanation is that DHEAS binds to the very low affinity receptors which are undetectable in ligand binding experiments. The most convincing evidence for the association of high affinity binding sites for [3H]PS and

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[3H]DHEAS with the GABAA receptors derives from pharmacological profiles of these neurosteroids, which are nonetheless distinct for PS and DHEAS. While [3H]PS binding to high affinity sites is inhibited primarily by picrotoxin, and minimally by barbiturates, the binding of [3H]DHEAS is robustly inhibited by barbiturates, but not by picrotoxin (Majewska et al., 1990a,b). These findings are consistent with the fact that PS, but not DHEAS, competitively inhibits the binding of [35S]TBPS or [3H]TBOB (which bind at the picrotoxin site of the GABAA receptor operated chloride ionophore) (Majewska and Schwartz, 1987; Majewska et al., 1990a). Hence, the pharmacological data suggest that the sites of action for PS may be the same or proximal to those for convulsants, whereas DHEAS may act at sites close or identical to those where barbiturates act at the GABA A receptor. Ligand binding experiments rendered further evidence of the distinction between sites of action of PS and DHEAS at the GABA A receptor. At micromolar concentrations PS exhibits GABAA-antagonistic features, but at nanomolar concentrations PS acts as an allosteric GABAA receptor agonist, because it potentiates [3H]GABA and [3H]benzodiazepine binding (Majewska et al., 1985; Majewska and Schwartz, 1987). DHEAS is devoid of these features; instead, it shifts dextrally the dose-response curve for pentobarbital to enhance binding of [3H]flunitrazepam (Majewska et al., 1990b). All together, these data suggest that while DHEAS acts primarily as an antagonist of the GABA A receptor, PS exhibits mixed GABA-agonistic/antagonistic features. 3.4.2. Neurosteroid binding sites: proteins or lipids? There were many attempts to identify membrane target sites of general anesthetics, which include the anesthetic steroids. Both lipid and protein theories received significant empirical support (Miller, 1986; Chiou et al., 1990). Analogous dilemma pertains to the discussion of the sites of steroid action on the GABAA receptors, because the chemical nature of steroids determines their affinity for both lipids and proteins. Our results from binding of radiolabeled neurosteroids to synaptosomal membranes suggest an interaction of neurosteroids with both lipids and proteins. As described above, the populations of high affinity [3H]PS and [3H]DHEAS binding sites in synaptosomal membranes appear to be associated with the GABA A receptors, but pharmacological binding profiles of both neurosteroids are distinct. In addition, PS and DHEAS binding sites have distinct sensitivity to protein-destructive treatments, such as thermal denaturation or proteolytic digestion. While PS binding seems resistant to both treatments (Majewska et al., 1990b), high affinity DHEAS binding sites are obliterated by both these treatments. However, treatment with phospholipase A2 destroys practically all binding of both PS and DHEAS to synaptosomal membranes (Demirgoren et al., 1991). Based on these observations, we have proposed that the high affinity PS binding sites could be the hydrophobic pockets of the GABA^ receptor protein that are deeply embedded in the neuronal membrane and thus, insensitive to protein digestion. Alterna-

tively, the PS binding site, may be the annular lipids intimately associated with the GABA A receptor. Theoretically, there should be one molecule of PS binding for each GABA A receptor, because densities of high affinity PS binding sites and those of GABAA receptors are the same. In contrast, the high affinity DHEAS binding sites seem to have a proteinaceous nature, although binding to these sites is also strongly dependent on the phospholipid milieu. 3.4.3. Evidence .from electrophysiology and receptor cloning

Electrophysiologicat experiments revealed that GABA-agonistic steroids exert their effects when applied to the neuron extracellularly, but not intracellularly, which implies that the location of steroid regulatory site is near the external surface of neuronal membrane (Lambert et al., 1990). Studies in molecular biology have provided interesting observations about the site of steroid action at the GABA A receptor. The mammalian GABAA receptors constitute a family of hetero-oligomeric receptors that express various combinations of polypeptide subunits (~,/~, Y, 6, e, and ~), encoded by different genes (Olsen and Tobin, 1990; Vicini, 1991). Various combinations of subunits (ether hetero- or homo-oligomeric) form ligand gated channels which differ in their sensitivity to GABA and benzodiazepines. Particularly, the expression of the benzodiazepine regulatory site at the GABAA receptor seems to require presence of the ~,2 subunit (Pritchett et al., 1989) in addition to earlier discovered ~ and/~ subunits (Schofield et al., 1987). One investigative team examined the effects of GABA-agonistic steroids on GABA-induced currents in human kidney embryonic cells, transfected with various combinations of cDNAs encoding the ~ 1,/~ 1, and y2 GABAA receptor subunits. They found that for every combination tested, the GABA-agonistic steroids potentiated GABA-induced currents, and PS blocked the currents (Puia et al., 1990a,b). These results suggested that the sites of steroid action are not associated with any particular subunit of the GABA A receptor, and that steroids act upon any type of chloride channel integral to the receptor. Somewhat different results were obtained by other groups of investigators, who tested inhibitory effects of PS on various cloned, bovine and human, subunits of GABAA receptor, expressed in Xenopus oocytes (Shingai et al., 1990). They used assorted combinations of • and/~, or ~,/~, and ~,, and observed that while PS inhibited GABA-induced currents in all subunit combinations, in the presence of ~ subunit the sensitivity of the receptor to PS was altered in different directions, depending on subunit combination. They also found that, PS (1-10 nM)--in about 25% of oocytes expressing combinations of ~2 or ~3 subunits--slightly enhanced the GABA-induced response (Shingai et al., 1990). The latter finding is the first electrophysiological evidence of the GABAagonistic properties of PS that corresponds with our biochemical observations, which showed an increase of GABA and benzodiazepine binding by nanomolar concentrations of PS (Majewska et al., 1985; Majewska and Schwartz, 1987). The observations of

MODULATORS OF THE G A B A A RECEPTOR

Shingai et al. (1990) suggest that GABAA receptors from different brain regions or neuronal compartments, which express different composition of the subunits, may distinctively respond to PS (and perhaps to other neurosteroids). This conclusion evidently contrasts with the observations of Puia et al. (1990b), thereby making apparent the need for further studies to clarify the nature of steroid action sites at the GABAA receptor. 3.4.4. Steroid regulatory sites at the G A B A a receptor: hypothetical model

The apparent pharmacological and biochemical heterogeneity of sites of steroid interaction with the GABA receptor suggests that the receptor protein may have several distinct regulatory sites for different steroids. Alternatively, steroids may be primarily binding to membrane phospholipids, with their functional groups interacting with the receptor and allosterically altering its function. Finally, steroids may be acting at the interphase of the receptor proteins and membrane phospholipids. Figure 2 illustrates a hypothetical model of interaction between the GABAA receptor and two neurosteroids: GABA-antagonistic PS, and GABAagonistic THP. The proposed model should be viewed as a heuristic concept, because currently the detailed information on this subject is not available. In the suggested model, the hydrophobic steroid backbone interacts with fatty acid chains of membrane phospholipids. The electro-negative atom at C-20 is present in both GABA-agonistic steroids, THP and THDOC, as well as in GABA-antagonistic, PS, thereby suggesting that this group may be essential for steroid binding to the GABAA receptor. On the other hand, 3ct-hydroxyl is vital for the expression of GABA-agonistie features of steroids, implying that this group may be responsible for the conformational alteration of the receptor structure, which opens the chloride channel. 3ct-Hydroxyl and oxygen at C-17 or C-20 may be interacting with the GABAA receptor protein via hydrogen bonds (Im et al., 1990), as is illustrated in the model.

385

The presence of sulfate at 3fl position seems to be important for the expression of GABA-antagonistic traits for both PS and DHEAS (Majewska et al., 1988, 1990b; Demirgoren et al., 1991). This highly polar sulfate group probably interacts with the membrane's surface, possibly with the phospholipid head groups. Because the actions of PS (Majewska et al., 1988) or DHEAS (Charles Spivak, personal communication) are not voltage dependent, these steroids probably do not act as open channel blockers. Mechanistically, it is conceivable that negatively charged steroid sulfate groups neutralize the cluster of positive charges located at the mouth of the chloride ionophore operated by the GABA A receptor (Schofield et al., 1987), thus reducing anion attraction and driving force. The GABA-antagonistic effects of PS and DHEAS could not be attributed to their nonspecific detergent-like actions, because other steroid sulfates (estratriol sulfate, cholesterol sulfate) were inactive, and detergents irreversibly blocked GABA-induced current, while the effects of DHEAS or PS were totally reversible (Majewska et al., 1988, 1990b; Demirgoren et al., 1991). Although steroid-modulation of ligand binding to GABAA receptor was demonstrated in purified receptor proteins (Olsen, 1990), the steroid interactions with annular lipids that are tightly bound to the GABA^ receptors cannot be ruled out. The concept of steroidal regulation of the GABAA receptor's function occurring by interaction with membrane lipids is suggested by the observation that anesthetic steroids stereoselectively alter the fluidity of pure lipid membranes, while the nonanesthetic analogs do not (Fesik and Makfiyannis, 1985).

4. BEHAVIORAL EFFECTS OF GABA-MODULATORY STEROIDS 4.1. GABA-AGONISTXCSTEROIDS The hypnotic and anesthetic features of certain steroids were recognized a long time ago when Cashin

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FIG. 2, Hypothetical model of interactions between steroids and the GABA^ receptor in the neuronal membrane. The model proposes the interaction of steroids at the interphase of the phospholipid membrane and receptor protein. Carbonyl at C-20 and hydroxyl at C-3~tinteract with the receptor by the formation of hydrogen bonds; whereas 3/)-sulfate interact with the external surface of the membrane by neutralizing a cloud of positive charges at the mouth of the receptor ionic channel.

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M.D. MAJEWSKA

and Moravek, in 1927, described the anesthetic action of intravenously injected cholesterol (Cashin and Moravek, 1927). Subsequently, Selye demonstrated rapid and reversible hypnotic actions of progesterone and deoxycorticosterone in the rat (Selye, 1942), and his studies eventually led to the development of steroidal anesthetics, which are structurally similar to the endogenous steroids, THP and THDOC (Gyermek and Soyka, 1975; Holzbauer, 1976). Although progesterone has been shown to potentiate electrophysiological responses to GABA in vivo (Smith et al., 1987), this hormone does not interact directly with the GABAA receptor (Majewska et al., 1986; Harrison et al., 1987); its actions are probably mediated via its reduced metabolite, THP. Hence, in light of current findings, the anesthetic-hypnotic effects of progesterone observed by Selye (Selye, 1942) can be explained by the potentiation of the GABAA receptor's function by its reduced metabolite, THP. Direct GABA-agonistic features of THP and THDOC also explain their hypnotic actions (Kraulis et al., 1975; Holzbauer, 1976; Holzbauer et al., 1985; Mendelson et al., 1987). Recently other than hypnotic behavioral effects of reduced metabolites of progesterone and deoxycorticosterone were also described, which can be explained by their GABA-agonistic features. For example, anxiolytic effects of THDOC, similar to those of benzodiazepines, were demonstrated in tests for anxiety in rodents (Crawley et al., 1986). THDOC was also tested in animal models for aggressive behaviors, and it was shown to reduce the level of defeat-induced analgesia, as well as to increase the tendency of rodents to undergo defeat in "resident-intruder" paradigms (Kavaliers, 1988). It is conceivable that the anxiolytic actions of THDOC during stress associated with agonistic encounters, mediate the propensity of animals to become defeated.

4.2. GABA-ANTAGONISTICSTEROIDS Hypnotic, anxiolytic and anesthetic actions of GABA-agonistic steroids, THP and THDOC, contrast with the behavioral effects of GABAantagonistic neurosteroid PS, which, when injected intracerebroventricularly (8/.tg/10/~l) reduced the pentobarbital-induced sleep time in rats (Majewska et al., 1989), but at lower doses PS tended to prolong the sleep time. The neurosteroid dehydroepiandrosterone (DHEA), when injected intraperitoneally in doses from 100 to 150mg/kg produced tonic-clonic seizures in mice, but in lower doses it caused behavioral sedation (Heuser et al., 1965). It is most likely that the convulsant effects of DHEA, which were counteracted by anesthetic steroids, were mediated via inhibition of GABA A receptors, because corresponding (high micromolar) concentrations of this steroid antagonize function of these receptors in brain synaptosomes and neurons (Majewska et al., 1990; Demirgoren et al., 1991). Figure 3 shows the structures of GABA-agonistic and -antagonistic neurosteroids and lists their neurochemical, electrophysiological and behavioral actions. 5. STEROIDS/GABA A RECEPTOR INTERACTIONS: PSYCHO-PHYSIO-PATHOLOGICAL ROLE

Ubiquitous GABA A receptors are present practically on every CNS neuron, and their function is to control neuronal excitation. Hence, precise regulation of activity of these receptors is vital for the expression of integrated brain functions. In this context, the bimodal modulation of GABAA receptors by neurosteroids may underlie numerous neurological and psychiatric events.

CH= I

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FIG. 3. Correlations of the biochemical, electrophysiological and behavioral actions of the GABAagonistic (THP and THDOC) and GABA-antagonistic (PS and DHEAS) neurosteroids.

MODULATORS OF THE

GABA^ RECEPTOR

387

It is very likely that the neurosteroids are secreted from oligodendrocytes into or near the synaptic cleft, where they alter neuronal excitability by modulating the function of GABAA receptors. When synaptic concentrations of PS or DHEAS increase in certain physiological or pathological situations, neuronal excitability and CNS arousal may be augmented. In contrast elevated synaptic concentrations of THP or THDOC should enhance neuronal inhibition mediated by GABA A receptors. In physiological conditions a fine interplay may exist between the hypnotic/anxiolytic steroids (THP, THDOC and androsterone) originating either in the CNS or in the periphery (Kraulis et al., 1975; Karavolas et al., 1984; Jung-Testas et al., 1989), and the GABA-antagonistic steroids (PS and DHEAS), produced mainly in the CNS (Baulieu et al., 1987; Hu et al., 1987). The two structurally distinct steroid groups not only counteract each other's actions at the GABAA receptor (Majewska and Schwartz, 1987; Majewska et al., 1988), but are also metabolically linked, as shown in Fig. 1.

GABA-agonistic stress steroids, THDOC and THP (Majewska et al., 1986), may be instrumental in the reinforcement of GABAA receptor function that has been observed with other stressors (Schwartz et al., 1987). The psychological reactions to environmental and internal stressors are complex, involving a multitude of interrelated biochemical and physiological events; some of which are beyond the scope of this article. Nevertheless, concentrating only on GABA-ergic steroids--whose levels increase during stress--one can easily discern a process in which the neuronal activity accompanying stress may be shaped by a counteracting interplay between the excitatory and inhibitory steroids. Consequently, it is conceivable that a diversity of individual stress reactions may be partially determined by the type of predominating steroids. It can also be foreseen that the behavioral reactions to stress would be markedly altered by changing plasma and CNS levels of neuroactive steroids, that accompany states such as puberty, aging, pregnancy, or phases of the menstrual cycle.

5.1. STRESS

5.2. DIURNALCYCLES

According to Selye, who pioneered studies on biological aspects of stress, stress reactions develop in the following stages: alarm reactions, resistance, and exhaustion (Selye, 1950). The stages of stress differ in their biochemical and physiological manifestations, including the adrenal secretion of catecholamines and steroids. Stress is accompanied by a massive secretion of adrenal steroids. The brain concentrations of the GABA-antagonistic neurosteroid, PS, were also reported to increase during stress (Baulieu et al., 1987). It is conceivable that the elevated CNS levels of PS, by suppression of inhibitory input of GABA, contribute to the heightened arousal which is characteristic of early stages of stress. In fact, PS may play an analeptic role in early stages of stress reactions. On the other hand, during stress ACTH stimulates adrenals to secrete THDOC and its precursor, deoxycorticosterone, in addition to glucocorticoids (Schambelan and Biglieri, 1972; Taylor et al., 1972). During swim stress increased levels of THP and THDOC were also recently measured in rat brain and plasma (Purdy et al., 1990). Because THP and THDOC have GABA-agonistic and anxiolytic features, they may protect the neurons from overstimulation. It is conceivable that these steroids come into play during later phases of stress reactions, being responsible for preservation of CNS homeostasis. Defeat-promoting actions of THDOC (Kavaliers, 1988) may also serve the purpose of self preservation during stress of aggressive encounters. Different acute stressors are known either to diminish (pain related stress; Biggio et al., 1987) or to potentiate (swim stress; Schwartz et al., 1987) the function of GABAA receptors. It is possible that both phenomena are mediated by stress steroids. For example, glucocorticoids and PS, at concentrations achieved during stress, reduce the density of the GABAA receptors (Majewska et al., 1985), and thus, may be responsible for the diminished activity of these receptors that has been observed with one type of stressor (Biggio et al., 1987). On the other hand,

Plasma levels of all adrenal steroid hormones undergo diurnal cyclical changes (Martin, 1985). Likewise, there are circadian variations in the brain concentrations of neurosteroids (Robel et al., 1987). For example, in the brain of the nocturnal animal rat, DHEA(S) and P(S) peak in the prenocturnal hours and their levels remain high through the first half of the dark cycle; while the lowest brain levels of these steroids occur in the early morning hours (Robel et al., 1987). The general pattern of cyclical variations of DHEA(S) levels in the rat brain resembles the pattern in plasma, and the crests of DHEA(S) and P(S) levels somewhat precede the peak of corticosterone both in the brain and plasma. In humans, the plasma level of DHEA(S) climaxes in the early morning hours, while its nadir takes place at the beginning of night (Hermida et al., 1985; Robel et al., 1987; Rosenfeld et al., 1971). It is most likely that the brain levels of DHA(S) in humans undergo similar diurnal changes as the levels in plasma (analogously to the rat). Because DHEA and DHEAS are antagonists of the GABA A receptor, these steroids at their peak levels in the brain may serve as physiological analeptics during periods of intense activity. 5.3. DEPRESSIONAND ANXIETY The role of GABA in the modulation of behavior and emotions and in the etiology of some affective disorders has been recently recognized (Berretini and Post, 1984). Depression and anxiety--which are related to stress with respect to numerous biological and psychological symptoms---seem to be associated with the inefficiency of GABA-ergic neurotransmission (Berretini and Post, 1984). GABA's function in depressive illness is suggested by the following observations: (1) GABA levels are low in the cerebrospinal fluid of depressed patients; (2) GABAagonists immediately inhibit depression; (3) intrahippocampal injections of bicuculline (GABA antagonist) produces helpless behavior in rats, while

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injections of GABA to this region immediately reverses helplessness; (4) tricyclic antidepressants inhibit GABA uptake and stimulate its release; (5) the antidepressant effects of monoamine oxidase (MAO) inhibitors can be blocked by bicuculline (Bertholini et al., 1986). The fact that GABA-mimetics enhance noradrenergic neurotransmission links classical monoaminergic, with more recent GABA-ergic, theory of depression. Finally, the usefulness of GABA-agonists in the treatment of anxiety and depression has been documented by clinical observers (Lloyd et al., 1983; Hollister et al., 1980). Anxiety and depression, like stress, are accompanied by hyperactivity of the hypothalamopituitary-adrenal axis (and hypoactivity of gonadal axis), which escalates the secretion of adrenal steroids. As the brain levels of neurosteroids, PS and DHEAS, are positively related with their adrenal secretion (Baulieu et al., 1987) and they are elevated during stress, one can predict that anxiety and depression are accompanied by increased in the brain levels of the excitatory neurosteroids. Hence, a role for the actions of neurosteroids at the GABA receptor as an underlying mechanism in mood disorders can be anticipated, and potential abnormalities in these actions should be examined as possible etiological factors in anxiety and depression disorders in humans.

5.4. AGGRESSION

Aggression includes a spectrum of different types of intrusive and offensive behaviors, with varying underlying mechanisms and psychosocial etiologies. Aggressiveness remains under strong hormonal control; especially androgens are known to induce intermale aggression in all species. Androgens were also shown to provoke infanticide by male rodents and castration or treatment with antiandrogenic drugs results in gentling of males of many species (Carlson, 1991). GABA-ergic mechanisms also seem to be involved in control of aggressiveness, thereby suggesting a role of neurosteroids in this behavior. In general, GABAagonistic drugs suppress aggressiveness. Injection of GABA-mimetics into the olfactory bulb of the rat inhibits "muricidal behavior", whereas injection of GABA-antagonists--induces it (Molina et al., 1986). Also, most benzodiazepines have a taming effect, although sometimes they may evoke paradoxical "rage reactions" and increased hostility in humans (Leventhal and Brodie, 1981). Consistent with the antiaggressive role of GABA-mimetics, GABAagonistic steroid, hydroxydione, and deoxycorticosterone (a precursor of THDOC) were shown to reduce aggressiveness (Kostowski et al., 1970). Also THDOC reduced aggression by promoting defeat of subordinate male mice engaged in agonistic confrontations (Kavaliers, 1988). Notwithstanding, the GABA-antagonistic neurosteroid, DHEA, was also shown to inhibit aggressiveness of castrated males against lactating females (Schlegel et al., 1985); thus demonstrating the need for more studies to evaluate the precise role of GABA and GABA-ergic steroids in control of different types of aggression.

5.5. PERSONALITYTRAITS

In recognition of the fact that various steroids influence neuronal excitability, and therefore CNS arousal, it is enticing to speculate that the profile of steroids in plasma and the CNS contributes to the manifestation of some personality traits. To a large extent this steroid profile is genetically determined, but it also changes during the developmental and physiological states such as puberty, aging, pregnancy or stress, and it is altered in some pathologies. Theoretically, a higher proportion of excitatory steroids to inhibitory ones would result in an emotionally unstable personality, such as is found in neurotic and anxious individuals. This personality is characterized by a higher level of resting arousal, a tendency to augment incoming stimuli, greater sensitivity to the environmental stimuli, a low threshold for positive hedonic tone, and higher levels of circulating cortisol (Eysenek, 1983). It is conceivable that elevated concentrations of GABA-antagonistic steroids, such as PS or DHEAS, may contribute to this "sensitive" type of personality by increasing CNS arousal. Elevated concentrations of GABA-antagonistic steroids may be responsible for, and typical for this personality, an inclination to develop depression (Eysenek, 1983) and high sedation threshold to barbiturates (Lader, 1983). In contrast, a higher proportion of inhibitory steroids to the excitatory ones could result in a more relaxed personality, with a tendency for blunted reaction to incoming stimuli. An extreme case of this personality would be a "sensation seeker", characterized by an atypically high threshold for arousal and requiring extremely strong stimuli to achieve positive hedonic tone. Characteristic for this personality type is also a tendency to criminal activities, and inclination to alcoholism and drug addiction. The concept of steroid involvement in personality traits is supported by the observations that humans, who are characterized by highly expansive personalities, have markedly lower plasma levels of the excitatory steroid, DHEAS, than the individuals with low expansivity (Hermida et al., 1985). 5.6. SEXUALFUNCTIONS,PREGNANCYAND "PosT-PARTUM BLUES"

GABA facilitates sexual receptivity in female rats. Because progesterone, deoxycorticosterone, and their reduced metabolites, promote lordosis, it was proposed that they act by potentiation of GABA-ergic inhibition in neurons involved in expression of this behavior (Schwartz-Giblin and Pfaff, 1987). Reduced metabolites of progesterone also participate in the control of gonadotropin secretion (Kubli-Garfias, 1984). The role of PS in the expression of male sexual behavior in rats is suggested by the fact that the amount of this neurosteroid is lowered in the olfactory bulbs of male rats after heterosexual exposure to females in estrus (Baulieu et aL, 1987). Because PS has GABA-antagonistic properties, the local change in its concentration may influence neuronal firing in the brain regions participating in male sexual behavior.

MODULATORSOF THE GABA A RECEPTOR

In many species, including humans, plasma and brain levels of THP and THDOC closely follow the levels of their precursors, progesterone and deoxycorticosterone (Ishikawa et al., 1974; Kraulis et al., 1975; Holzbauer, 1976; Rosciszewska et al., 1986; Purdy et al., 1990a). For example, during pregnancy in mammals, the maternal plasma concentrations of THP and THDOC are high due to the extremely high levels of their precursor hormones, progesterone and deoxycorticosterone, and due to increased activity of their synthesizing enzymes in the placenta and fetal tissues (Milewich et al., 1979, 1987). In pregnancy, the brain concentrations of THP and THDOC are also elevated, which may potentiate the function of GABAA receptors (Backstrom et al., 1990). Indeed, we observed that the affinity of GABAA receptors in the brains of pregnant rats was markedly increased as if these receptors were influenced by GABA-agonistic steroids (Majewska et al., 1989). The post-partum period was associated with a drastic decrease of the density of GABA Areceptors, which could result from exposure to high levels of glucocorticoids or PS (Majewska et al., 1985) during parturition. Similar changes to those described above may occur in humans, contributing to alterations of mood and psyche, such as the feeling of well being or depression, typifying human pregnancy and puerperium, respectively. During pregnancy high levels of the anxiolytic-hypnotic steroid, THP, may also contribute to increased somnolence, which is characteristic of pregnant women. On the other hand, the anxiety and depression associated with the post-parturn period may represent physiological withdrawal from several-month exposure to the endogenous anxiolytics. Analogous steroid-mediated modifications of GABAA receptors may occur during the menstrual cycle. High levels of reduced metabolites of pro= gesterone during luteal phase (Rosciszewska et al., 1986) may result in development of autodependency on this natural anxiolytic, which, when followed by withdrawal during the premenstrual phase, may contribute to symptoms of premenstrual tension. 5.7. SEIZURES Impairment of GABA-ergic neurotransmission is linked to seizure disorders, and frequency of seizures is altered in physiological states such as stress and pregnancy, in which the secretion of steroid hormones is altered. Since anesthetic steroids increase seizure threshold, while convulsant steroids lower it (Fidgor et al., 1957; Heuser et al., 1961; Gyermek et al., 1968), one can predict that the profile of circulating steroids would affect occurrence of seizures. Increased exposure to anesthetic steroids during pregnancy or luteal phase may underlie the reduced incidence of seizures, typically associated with these states (Rosciszewska et al., 1986; Baekstrom et al., 1990). Conversely, premenstrual reduction of progesterone and its metabolites level may contribute to the observed increased frequency and strength of seizures (Rosciszewska et al., 1986; Backstrom et al., 1990).

389

5.8. FEEDING AND BLOOD PRESSUREREGULATION

GABA, acting primarily via hypothalamic GABAA receptors, influences feeding behavior. Microinjection of GABAA agonistic drugs (muscimol, benzodiazepines, barbiturates) into the ventromedial hypothalamus induces feeding by inhibiting the satiety center (Matsumoto, 1989). Therefore, one can predict that steroids with GABA-agonistic features would increase feeding by potentiating function of hypothalamic GABA A receptors. This concept remains to be tested experimentally, but such a mechanism could explain the increased appetite and food consumption occurring during pregnancy and the luteal phase of the menstrual cycle, when high levels of GABAagonistic steroids are present. A role of GABA-enhancing steroids in the regulation of cardiovascular function has been suggested by the positive correlation between hypertension and reduced adrenal secretion of THP (Holzbauer et al., 1985). The presumed hypotensive effects of this steroid may be both central and peripheral in nature, via potentiation of G A B A A receptors in neural and vascular tissues which regulate heart rate and blood pressure. 5.9. COGNITIVE FUNCTIONS The role of GABA in learning and memory is suggested by the finding that GABA antagonists facilitate the induction of long term potentiation (LTP) in the hippocampus (Wigstrom and Gustaffson, 1985). LTP is the neurophysiological property mediated via the actions of excitatory neurotransmitters and believed to be fundamental for long-term memory. An inhibitory input provided by the local GABA-ergic interneurons probably sets a threshold for postsynaptic modification of excitatory inputs (Douglas et al., 1982). Because neurosteroids regulate function of GABA, receptors it is likely that they affect cognitive functions. PS, DHEAS and DHEA, due to their GABAantagonistic freatures, probably potentiate long-term memory, whereas GABA-agonistic steroids, most likely impair memory processes. These predictions are justified by the fact that hypnotics (barbiturates, benzodiazepines) produce amnesia (Roehrs et al., 1983), whereas stimulants (picrotoxin, pentylenetetrazol) reverse amnesia and improve memory (Breen and McGaugh, 1961; Irwin and Benuazizi, 1966). Supporting this concept is the finding that intravenous injections of the reduced metabolite of corticosterone, 5~t-dihydrocorticosterone, impairs development of LTP in the rat (Dubrovsky et al., 1987). Although this steroid by itself is devoid of clear GABA-agonistic features it could be metabolized in vivo to the steroid(s), which possess such qualities, and could inhibit the LTP by potentiation of the inhibitory function of GABA A receptors. The empirical evidence supports these predictions, as the excitatory steroids DHEAS and DHEA have been shown to improve memory in aging mice (Flood and Roberts, 1988a) and to prevent pharmacologically induced amnesia in mice (Flood et al., 1988b). The role of progesterone and its reduced metabolites, in cognitive functions is suggested by the finding that

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M.D. MAJEWSKA

cyclical variations in cognitive performance occur in some women (Sanders and Reinisch, 1985). In the high-estrogen preovulatory phase, the automatized memory tasks, such as speed of reading and talking are facilitated, but they are impaired during the high-progesterone luteal phase. The reverse is true for perceptual-reconstructing tasks, which require inhibition of automatized responses, and which improve during luteal phase. The mechanisms of these phenomena are not known, but because progesterone suppresses the glutamate-induced neuronal excitation (Smith et al., 1987) it is likely that these phenomena are involved in cyclicity of cognitive functions. THP may also affect cognitive functioning, because it inhibits the glutamate-induced depolarization by activating G A B A A receptors (Lambert et aL, 1990). It is likely that non-GABA-ergic mechanisms also participate in steroid effects on cognition; nonetheless, the GABA A receptors, being targets for neurosteroid actions, seem to play a crucial role in these phenomena. Figure 4 is a graphical presentation of the neural actions of neurosteroids and their possible physiological effects. 5.10. DEFECTSIN STEROIDOGENESIS Because endogenous steroids bidirectionally modulate function of the GABAA receptor and affect neuronal excitability, aberrant steroid synthesis

could form the basis for some CNS disorders. For example, Cushing's disease, characterized by oversecretion of adrenal steroids, is linked to severe neuropsychiatric manifestations, including mood lability, insomnia, agitation, and others, that resemble symptoms of depression. Adrenal insufficiency in Addison's disease is also accompanied by psychiatric disturbances, such as irritability, apathy, fatigue, somnolence, dulling of intellect, and memory deficits. Some of these psychoneurological manifestations may result from abnormal steroid-GABA receptor interactions. In addition to gross defects in steroid synthesis, the physiologically important steroid balance may be disturbed by errors of individual enzymes in steroidogenic organs or in the CNS. The biosynthesis of neurosteroids may also be influenced by diet or various psychotropic drugs. For example, brain activity of 5ct-steroid-reductase, the key enzyme responsible for conversion of progesterone to THP, can be markedly increased by the administration of hypnotic and anticonvulsant drugs such as phenytoin, barbiturates, diazepam, or carbamazepine, as well as by psychostimulants such as caffeine or methamphetamine (Kaneyuki et al., 1979). In contrast, a 5-week dietary deficiency of riboflavin resulted in complete disappearance of 5~t-steroid reductase in the brain (Bertics and Karavolas, 1984). These results support the conclusion that various pharmacological or dietary treatments may alter the central synthesis of neurosteroids, and thereby, profoundly influence the excitability of neurons.

6. SUMMARY

FIG. 4. Illustration of the neuromodulatory actions of neurosteroids and the physiological functions, in which this modulation may come into play. In the model, PS and DHEAS inhibit function of neuronal GABAA receptors. This inhibitory effect may contribute to increases in anxiety and arousal, and enhancement of memory processes. THP and THDOC potentiate function of neuronal GABAA receptors, and thus may reduce anxiety and aggression, while potentiating eating, sleep and sexual behavior.

The abundant CNS cholesterol and its sulfate derivative serve as precursors of different neurosteroids, which bidirectionally modulate neuronal excitability, by potentiating or inhibiting function of the GABA A receptors. The regulation of G A B A A receptors in the CNS by the steroids of central or peripheral origin may constitute a vital means of brain-body communication, essential for integrated whole organism responses to external stimuli or internal signals. Modulation of the brain GABA receptors by neurosteroids may form the basis of a myriad of psychophysiological phenomena, such as memory, stress, anxiety, sleep, depression, seizures and others. Therefore, the aberrant synthesis of centrally-active steroids may contribute to defects in neurotransmission, resulting in a variety of neural and affective disorders. The biosynthesis of neurosteroids may also be altered by diet and certain psychotropic drugs, thereby affecting excitation of neurons. Hereditary differences in the level of synthesis and catabolism of different neurosteroids may underlie individual variations in CNS excitability, contributing to differences in personality traits, including the inherited susceptibility to drug addiction. Acknowledgements--I am grateful to all my collaborators for their contributions at various stages of my studies on neurosteroids and to Dr Linda Weinhold for critical reading of this manuscript.

MODULATORSOF THE GABA A RECEPTOR

REFERENCES BACKSTROM,T., GEE, K. W., LAN, N., SORENSEN,M. and WAHLSTROM,G. (1990) Steroids in relation to epilepsy and anestheisa. In: Steroids and Neuronal Activity, pp. 225-230. Ciba Foundation Symposium, 153. Wiley: Chichester. BARKER,J. L., HARRISON,LANGE,N. L., MAJEWSKA,M. D. and OWEN, D. G. (1986) Voltage-clamp studies of the potentiation of the GABA-activated chloride conductance by alphaxalone and reduced metabolite of progesterone. J. Physiol., Lond. 377, 83P. BAULmU,E.-E. and ROBEL,P. (1990) Neurosteroids: A new brain function. J. Steroid Biochem. molec. Biol. 37, 395~t03. BAULIEU, E.-E., ROBEL, P., VATIER, O., HAUG, A., LE GOASCOGNE,C. and BOORREAU,E. (1987) Neurosteroids: pregnenolone and dehydroepiandrosterone in the rat brain. In: Receptor-Receptor Interaction, A New Intramembrane Integrative Mechanism, pp. 89-104. Eds. K. FUXE and L. F. AGNATI. MacMillan: Basingstoke. BERRETINI,W. H. and POST, R. M. (1984) GABA in affective illness. In: Neurobiology o f Mood Disorders, pp. 673-685. Eds. R. M. POST and J. C. BALLANGER. Williams & Wilkins: Baltimore. BERTHOLINI,G., SCATTON,B., ZIVKOVITZ,B. and LLOYD, K. G. (1986) On the mode of antidepressant action of GABA receptor agonist and monoamine-uptake inhibitors. In: GABA and Mood Disorders, pp. 105-112. Eds. G. BARTHOLINIet al. Raven Press: New York. BERTICS,P. J. and KARAVOLAS,H. J. (1984) Partial characterization of microsomal and solubilized hypothalamic progesterone 5c~-reductase. J. Steroid Biochem. 21, 305-314. B1GGIO,G., CONCAS,A., MELE, S. and COROA,M. G. (1987) Changes in GABAergic transmission induced by stress, anxiogenic and anxiolytic/%carbolines. Brain Res. Bull. 19, 301-308. BREEN, R. A. and McGAUGH, J. L. (1961) Facilitation of maze learning with post-trial injections of picrotoxin. J. comp. Physiol. Psychol. 54, 498-501. CANICK,J. A., VACCARO,D. E., LIVINGSTON,E. M., LEEMAN, S. E., RYAN, K. J. and Fox, T. O. (1986) Localization of aromatase and 5a-reductase to neuronal and nonneuronal cells in the fetal rat hypothalamus. Brain Res. 372, 277-282. CARETTE, B. and POULAIN,P. (1984) Excitatory effect of dehydroepiandrosterone, its sulphate esther and pregnenolone sulphate, applied by iontophoresis and pressure, on single neurons in the septo-optic area of the guinea pig. Neurosci. Lett. 45, 205-210. CARLSON,N. R. (1991) Aggressive Behavior. In: Physiology o f Behavior, pp. 357-363. Allyn and Bacon: Boston. CASl-nN, M. F. and MORAVEK,V. (1927) The physiological actions of cholesterol. Am. J. Physiol. 82, 294-298. CELOTTI,F., MELCANGI,R. C., NEGRI-CESl,P. and BALLABIO, M. (1987) Differential distribution of the 5c~-reductase in the central nervous system of the rat and the mouse: are the white matter structures of the brain target tissue for testosterone action? J. Steroid Biochem. 26, 125-129. CHIOU, J.-S., MA, S.-M., KAMAYA,H. and UEDA,I. (1990) Anethesia cutoff phenomenon: interfacial hydrogen bonding. Science 248, 583-585. CORPECHOT, C., ROBEL, P., AXELSON,M., SJOVALL,J. and BAULIEU,E.-E. (1981) Characterization and measurement of dehydroepiandrosterone sulfate in rat brain. Proc. natn. Acad. Sci. U.S.A. 78, 4704-4707. CORPECHOT,C., SYNGUELAKIS,M., TALHA,S., AXELSON,M., SJOVALL, J., VIHKO, R., BAULmU, E.-E. and ROBEL, P. (1983) Pregnenolone and its sulfate ester in the rat brain. Brain Res. 270, 119-125. CRAWLEY,J. N., GLOWA,J. R., MAJEWSKA,M. D. and PAUL, S. M. (1986) Anxiolytic activity of endogenous adrenal steroid. Brain Res. 339, 382-386.

391

DEMIRGOREN,S., MAJEWSKA,M. D., SPlVAK, CH.E. and LONDON, E. D. (1991) Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor. Neuroscience, 45, 127-135. DOUGLAS, R. M., GODDARD,G. V. and RIVES, M. (1982) Inhibitory modulation of long-term potentiation: evidence for a postsynaptic control. Brain Res. 240, 259-272. DUBROVSKY, B. O., LIQUORNIK, M. S., NOBLE, P. and GIJSBERS,K. (1987) Effects of 5~,-dihydrocorticosterone on evoked responses and long-term potentiation. Brain Res. Bull. 19, 635-638. ENNA,S. W. J. and KARm)N,E. W. (1986) GABAA receptors: an overview. In: Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 241-259. Eds. R. W. OLSEN and J. C. VENTER. Liss: New York. EYSENEK, H. J. (1983) Psychophysiology and personality: extraversion, neuroticism and psychoticism. In: Physiological Correlates o f Human Behaviour, pp. 13-29. Academic Press: London. FESIK, S. W. and MAKRIYANNIS,A. (1985) Geometric requirements for membrane perturbation and anesthetic activity, Conformational analysis of alphaxalone and ~6-alphaxalone and 2H NMR studies on their interactions with model membranes. Molec. Pharmac. 27, 624--629. FIDGOR,S. K., KODET,M. J., BLOOM,B. M., ANGELLO,W. J., P'AN, S. Y. and LAUBACtt,G. D. (1957) Central activity and structure in a series of water soluble steroids. J. Pharmac. exp. Ther. 119, 299-309. FLOOD, J. F. and ROBERTS,E. (1988a) Dehydroepiandrosterone sulfate improves memory in aging mice. Brain Res. 448, 178-181. FLOOD, J. F., SMITH, G. E. and ROBERTS,E. (1988b) Dehydroepiandrosterone and its sulfate enhance memory retention in mice. Brain Res. 447, 269-278. GEE, K. W., BOLGER,M. B., BRINTON,R. E., CORINI,H, and McEWEN, B. S. (1988) Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action. J. Pharmac. exp. Ther. 246, 803-812. GYERMEK, L. and SOYKA,L. F. (1975) Steroid anesthetics. Anesthesiology 42, 331-344. GYELMEK, L., IRmRTE, J. and OL~,BBE,P. (1968) Steroids. CCCX. Structure-activity relationship of some steroidal hypnotic agents. J. reed. Chem, 11, 117-125. HARRISON,N. L. and SIMMONDS,M. A. (1984) Modulation of GABA receptor complex by a steroid anesthetic. Brain Res. 323, 284-293. HARRISON,N. L., MAJEWSKA,M. D., HARRINGTON,J. W. and BARKER,J. L. (1987) Structure--activity relationships for steroid interaction with the y-aminobutyric acidA receptor complex. J. Pharmac. exp. Ther. 241) 346--353. HERMIDA, R. C., HALBERG,F. and DEL Pozo, F. (1985) Chronobiologic pattern discrimination of plasma hormone, notably DHEAS and TSH, classifies an expansive personality. Chronobiologia 12, 105-136. HEUSER,G. and EIDELBERG,E. (1961) Steroid induced convulsions in experimental animals. Endocrinology 69, 915-924. HOLLiSTER,L. E., GREENBLATT,D. J., RICKELS,K., AYD, F. J. and GREIrCER,G. E. (1980) Benzodiazepines: current update. Psychosomatics 21 (Suppl.), 1-32. HOLZBAUER, M. (1976) Physiological aspects of steroids with anaesthetic properties. Med. BioL 54, 227-242. HOLZBAUER,M., BIRMINGHAM,M., DE NICOLE,A. F. D. and OLIWZ, J. T. (1985) In vivo secretion of 3~-hydroxy-5~pregnane-20-one a potent anesthetic steroid by adrenal gland in rat. jr.Steroid Biochem. 22, 97-102. HU, Z.-Y., BOURKEAU, E., JtmG-TEsTAS, I., ROBEL, P. and BAULmU, E.-E. (1987) Neurosteroids: Oligodendrocyte mitochondria convert cholesterol to pregnenolone. Proc. nam. Acad. Sci. U.S.A. 84, 8215-8215.

392

M.D. MAJEWSKA

IM, W. B., BLAKEMAN,D. P., DAVIS, J. P. and AVER, D. E. (1990) Studies on mechanism of interactions between anesthetic steroids and y-aminobutyric acid A receptors. J. Pharmac. exp. Ther. 37, 429--434. IRWIN, S. and BENUAZIZI,A. (1966) Pentylenetetrazol enhances memory function. Science 152, I00-102. ISHIKAWA,S., SAWAOA,T., NAKAMURA,Y. and MARIOKA,T. (1974) Ovarian secretion of pregnane compounds during estrous cycle and pregnancy in rats. Endocrinology 94, 1615-1620. IWAMORI, M., MOSER, H. W. and KISHIMOTO, Y. (1976a) Cholesterol sulfate in rat tissues. Tissue distribution, developmental changes and brain subcellular localization. Biochem. biophys. Acta 441, 268-279. IWAMORI, M., MOSER, H. W. and KISHIMOTO, Y. (1976b) Steroid sulfatase in brain: comparison of sulfohydrolase activities for various steroid sulfates in normal and pathological brains, including various forms of metachromatic leukodystrophy. J. Neurochem. 27, 1389-1395. JUNG-TESTAS, 1., HU, Z. Y., BAULIEU, E.-E. and ROBEL, P. (1989a) Steroid synthesis in rat brain cell cultures. J. Steroid Biochem. 34, 511-519. JUNG-TESTAS, 1., HU, Z. H., BAULIEU, E.-E. and ROBEL, P. (1989b) Neurosteroids: Biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells. Endocrinology 125, 2083-2091. KANEYUKI, T., KOHSAKA, M. and SHOHMORI, T. (1979) Sex hormone metabolism in the brain: influence of central acting drugs on 5or-reduction in rat diencephalon. Endocr. Jap. 26, 345-351. KARAVOLAS,H. J., BERTICS,P. J., HODGES,n . and RUDIE, N. (1984) Progesterone processing by neuroendocrine structures, ln: Metabolism of Hormonal Steroids, pp. 149-169. Eds. F. CELOTTI et al. Raven Press: New York. KAVALIERS,M. (1988) Inhibitory influences of adrenal steroid, 3ct,5ct-tetrahydrodeoxycorticosterone on aggression and defeat-induced analgesia in mice. Psychopharmac., Berlin 95, 488-492. KOSTOWSKI, W., REWERSKI, W. and PIECHOCKI, T. (1970) Effects of some steroids on aggressive behaviour in mice and rats. Neuroendocrinology 6, 311-318. KRAULIS, I., FOLDES, G., TRAIKOV,H., DUBROVSKY,B. and BIRMINGHAM,M. K. (1975) Distribution, metabolism and biological activity of deoxycorticosterone in the ventral nervous system. Brain Res. 88, 1-14. KRAUSE, J. E. and KARAVOLAS, H. J. (1980) Subcellular localization of hypothalamic progesterone metabolizing enzymes and evidence for distinct NADH- and NADPH-linked 3~t-hydroxysteroid oxidoreductase activities. J. Steroid Biochem. 13, 271-280. KRIEGER, N. R. and SCOTT, R. G. (1989) Nonneuronal localization for steroid converting enzyme: 3~t-hydroxysteroid oxidoreductase in olfactory tubercle of rat brain. J. Neurochem. 52, 1866-1870. KUBLI-GARFIAS, C. (1984) Physiological role of 5~- and 5fl-progesterone metabolites on the CNS. TIPS October, 439-441. LACROIX, C., FIET, J., BENAIS,J.-P., GUEUX, B., BONETE, R., VILLETTE, J.-M., GOORMEL, B. and DREUX, C. (1987) Simultaneous radioimmunoassay of progesterone, androst-4-ene-dione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone in specific regions of human brain. J. Steroid Biochem. 28, 317-325. LAOER, M. (1983) Anxiety and depression. In: Physiological Correlates of Human Behaviour, pp. 155-167. Eds. A. GALE and J. A. EDWARDS. Academic Press: London. LAMBERT,J. J., PETERS,J. A. and COTTRELL (1987) Actions of synthetic and endogenous steroids on the GABAA receptor. TIPS 81, 224-227. LAMBERT, J. J., PETERS,J. A., STRUGGES, N. C. and HALES, T. G. (1990) Steroid modulation of the GABAA receptor complex: electrophysiological studies. In: Steroids and

Neuronal Activity, pp. 56-70, Ciba Foundation Symposium 153. Wiley: Chichester. LANTHEIR, A. and PATWARDHAN,V. V. (1986) Sex steroids and 5-en-3fl-hydroxysteroids in specific regions of the human brain and cranial nerves. J. Steroid Biochem. 25, 445-449. LE GOASCOGNE,C., ROBEL, P., GOUEZOU, M., SANANES,N., BAULmU,E.-E. and WATERMAN,M. (1987) Neurosteroids: Cytochrome P-450~ in rat brain. Science 237, 1212-1214. LEVENTHAL,B. and BROmE, H. K. (198 I) The pharmacology of violence. In: Biobehavioral Aspects of Aggression, pp. 85-106. Eds. D. A. HAMBURG and M. B. TRUDEAU. Liss: New York. LmBERMAN,S., GREENFIELD,N. J. and WOLFSON,A. (1984) A heuristic proposal for understanding steroidogenic processes. Endocr. Rev. 5, 128-148. LLOYD, K. G., MORSELLI, P. L., DEPOORTERE, H., FOURNIER, V., ZIVKOVIC,B., SCATTON, B., BROEKKAMP,C., WORMS,P. and BERTHOLINI,G. (1983) The potential use of GABA agonists in psychiatric disorders: Evidence from studies with progabide in animal models and clinical trials. Pharmac. Biochem. Behav. 18, 957-966. MAJEWSKA, M. D. (1987a) Steroids and brain activity. Essential dialogue between body and mind. Biochem. Pharmac. 36, 3781-3788. MAJEWSKA, M. O. (1987b) Antagonist-type interaction of glucocorticoids with the GABA receptor-coupled chloride channel. Brain Res. 418, 377-382. MAJEWSKA, M. D. (1988) Interaction of ethanol with the GABA a receptor in the rat brain: possible involvement of endogenous steroids. Alcohol 5, 269-273. MAJEWSKA, M. D. (1990) Steroid regulation of the GABA A receptor: ligand binding, chloride transport and behaviour. In: Steroids and Neuronal Activity, pp. 83-96. Ciba Foundation Symposium 153. Wiley: Chichester. MAJEWSKA, M. D. and SCHWARTZ, R. D. (1987) Pregnenolone-sulfate: an endogenous antagonist of the yaminobutyric acid receptor complex in brain? Brain Res. 404, 355-360. MAJEWSKA, M. D., BISSERRE,J. C. and ESKAY, R. E. (1985) Glucocorticoids are modulators of GABA A receptors in brain. Brain Res. 339, 178-182. MAJEWSKA, M. D., HARRISON, N. L., SCHWARTZ, R. D., BARKER, J. L. and PAUL, S. M. (1986) Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232, 1004-1007. MAJEWSKA, M. D., MmNVmLE, J. M. and VICINI, S. (1988) Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons. Neurosci. Lett. 90, 279-284. MAJEWSKA, M. D., BLUET-PAJoT, M.-T., PxOBEL, P. and BAULmU, E.-E. (1989) Pregnenolone sulfate antagonizes barbiturate-induced sleep. Pharmac. Biochem. Behav. 33, 701-703. MAJEWSKA, M. D., FORD-RICE, F. and FALKAY, G. (1989) Pregnancy-induced alterations of GABA^ receptor sensitivity in maternal brain: an antecedent of post-partum 'blues'? Brain Res. 482, 397-401. MAJEWSKA, M. D., DEMIRGOREN, S. and LONDON, E. D. (1990a) Binding of pregnenolone sulfate to rat brain membranes suggests multiple sites of steroid action at the GABA A receptor. Fur. J. Pharmac.--molec. Pharmac. Section 189, 307-315. MAJEWSKA, M. D., DEMIRGOREN, S., SPIVAK, CH. E. and LONDON, E. D. (1990b) The neurosteroid dehydroepiandrosterone sulfate is an antagonist of the GABA A receptor. Brain Res. 526, 143-146. MARTIN, C. (1985) Hormone secretion cycles. In: Endocrine Physiology, pp. 62-65. Oxford University Press: New York. MATSUMOTO, R. R. (1989) GABA receptors: are cellular differences reflected in function? Brain Res. Bull. 14, 203-225.

MODULATORSOF THE GABAA RECEPTOR McEWEN, B. S. (1982) Glucocorticoids and Hippocampus: Receptors in search of function. In: Adrenal Actions on Brain, pp. 1-22. Eds. D. GArClXNand D. PFAFF. Springer: Berlin. McEWEN, B. S., DAVIS,P. G., PARSONS,B. and PFAFF,D. W. (1979) The brain as a target for steroid hormone action. A. Rev. Neurosci. 2, 65-112. MENDELSON,W. B., MARTIN,J. V., PERLIS,M., WAGNER,R., MAJEWSKA,M. D. and PAUL,S. i . (1987) Sleep induction by adrenal steroid in the rat. Psychopharmac., Berlin 93, 226-229. MIENVlLLE,J.-M. and VICINI,S. (1989) Pregnenolone sulfate antagonizes GABA A receptor-mediated currents via a reduction of channel opening frequency. Brain Res. 489, 190-194. MILEWICH, L., GANT, N. F., SCHWARTZ,B. E., CHEN, G. T. and McDONALD, P. C. (1979) 5~-Reductase activity in human placenta. Am. J. Obstet. Gynecol. 133, 611-617. MILEWICH, L., KAIMAL,V. and JOHNSON,A. R. (1987) Steroid 5~-reductase activity in endothelial cells from human umbilical cord vessels. J. Steroid Biochem. 26, 561-567. MILLER,K. (1986) Are lipids or proteins the target of general anesthetic action? TINS 6, 49-51. MOLINA, V., CIESIELSKI,L., GOBAILLE,S. and MANDEL,P. (1986) Effects of potentiation of the GABAergic neurotransmission in the olfactory bulbs on mouse-killing behaviour. Pharmac. Biochem. Behav. 24, 657-664. OLSEN, R. W. (1990) In: Steroids and Neuronal Activity, pp. 103-104. Ciba Foundation Symposium 153. Wiley: Chichester. OLSEN, R. W. and TOmN, A. J. (1990) Molecular biology of GABAA receptors. FASEB J. 4, 1469-1480. ONG, J., KERR, D. I. B. and JOHNSTON,G. A. R. (1987) Cortisol: a potent biphasic modulator at GABAA-receptor complexes in guinea pig isolated ileum. Neurosci. Lett. 82, 101-106. PARSONS,B. and PFAFF,D. (1985) Progesterone receptors in CNS correlated with reproductive behavior. In: Actions of Progesterone on the Brain, pp. 103-140. Eds. D. GANTEN and D. PFAFF. Springer: Berlin. PETERS, J. A., K1RKNESS,E. F., CALLACHAN,H., LAMBERT, J. J. and TURNER,A. J. (1988) Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids. Br. J. Pharmac. 94, 1257-1269. PRITCHETT,D. B., SONTHE]~mR,H., StaYERS,B. D., YMER,S., KETTENMAN,H., SCHOFIELD,P. R. and SEEnURG,P. H. (1989) Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338, 582-585. POIA,G., SANTI,M. R., VICINI,S., PRITCHETT,D. B., PURDY, R. H., PAUL,S. M., SEEBURG,P. H. and COSTA,E. (1990a) Neurosteroids act on recombinant human GABAA receptors. Neuron 4, 759-765. PUXA,G., SANTI,M. R., VIClNI,S., SEEBURG,P. H. and COSTA, E. (1990b) Differences on receptor structure requirement for neurosteroid and benzodiazepine facilitation of GABA-fated currents. Soc. Neurosci. Abstr. 16, 804. PusvY, R., MOORE, P. H. JR, RAO, N., HAGINO, N., YAMAGUCHI,T., SCHMIDT,P., RUSINOW,D. R., MORROW, A. L. and PAUL, S. M. (1990a) Radioimmunoassay of 3a-hydroxy-5,,-pregnan-20-one in rat and human plasma. Steroids 55, 290-295. PURDY, R. H., MoRRow, A. U, MOORE,P. H. JR and PAUL, S. M. (1990b) Stress-induced elevation of GABA^ receptor-active 3,,-hydrohysteroids in the rat brain. Soc, Neurosci. Abstr. 16, 691. PIKER, W. F., JR, BAKER,T. and SmTRE, A. (1982) Electrophysiologic and clinical aspects of glucocorticoids on certain neural systems. In: Adrenal Actions on Brain, pp. 69-106. Eds D. GANTEN and D. PFAFF. Springer: Berlin. RDBEL, P., BAULIEU,E.-E, SYNOtmLAIaS,M. and HALBI~O, F. (1987) Chronobiologlc dynamics of 5-~-hydroxy. steroids and glucocorticoids in rat brain and plasma and

393

human plasma. In: Advances in Chronobiology, Part A., pp. 451-465. Ed. D. K. HAYESet al. Wiley: New York. RoEtmS, T., ZORICK,F. J., SICKLESTEEL,J. M., WITTIG,R. M., HARTSE,K. M. and ROTH, T. (1983) Effects of hypnotics on memory. J. Clin. Psychopharmac. 3, 310-313. ROMMERTS, F. F. G. and VAN DER MOLEN, H. J. (1971) Occurrence and localization of 5u-steroid reductase, 3uand 17/}-hydroxysteroid dehydrogenases in hypothalamus and other brain tissues of the male rat. Biochem. biophys. Acta 248, 489-502. ROSCISZEWSKA,D., BUNTNER,B., Guz, I. and ZAWlSZA,L. (1986) Ovarian hormones, anticonvulsant drugs, and seizures during the menstrual cycle in women with epilepsy. J. Neurol. Neurosurg. Psychiat. 49, 47-51. ROSELLI, CH.E. and SNIPESCH. A. (1984) Progesterone 5~(-reductase in mouse brain. Brain Res. 305, 197-202. ROSENFELD,R. S., HELLMAN,L., ROFFWARG,H., WEITZMAN, E. D., KUKUSHIMA,D. K. and GALLAGHER,T. F. (1971) Dehydroepiandrosterone is secreted episodically and synchronously with cortisol by normal man. J. clin. Endocr. 33, 87-92. ROUSSEAU,G. G. and BAXTER,J. D. (1979) Glucocorticoids and metabolic code. In: Glucocorticoid Hormone Actions, pp. 613-629. Eds. J. D. BAXTER and G. G. ROUSSEAU. Springer: Berlin. SANDERS,S. A. and RE1NlSCH,J. M. (1985) Behavioral effects on humans of progesterone related compounds during development and in the adult. In: Actions of Progesterone on the Brain, Eds. D. GANTENand D. PEAFF. pp. 175--206. Springer: Berlin. SCHAMeELAN,M. and BmLmRI, E. G. (1972) Deoxycorticosterone production and regulation in man. J. din. Endocr. Metab. 34, 695-6-699. SCHLEGEL, M. L., SPETZ, J. F., ROBEL, P. and HAUG, M. (1985) Studies on the effects of dehydroepiandrosterone and its metabolites on attack by castrated mine on lactating intruders. Physiol. Behav. 34, 867-870. SCHOFIELD,P. R., DARLISON,M. G., FUJITA,N., BURT,D. R., STEPHENSON, F. A., RODRIGUEZ, H., RIME, L. M., RAMACHANDRAN, J., REALE, V., GLENCORSE, T. A., SEEBURG, P. H. and BARNARO,E. A. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328, 221-227. SCHWARTZ-GIBLIN,S. and PFAFF, D. W. (1987) Commentary, Integrative Psychiat. 5, 258-273. SCHWARTZ,R. D., WEmS,M. G., LABARCA,R., SKOLNICK,P. and PAUL,S. M. (1987) Acute stress enhances the activity of the GABA receptor-gated chloride ion channel in brain. Brain Res. 411, 151-155. SELYE,H. (1942) Correlations between the chemical structure and the pharmacological actions of the steroids. Endocrinology 30, 437-452. SELYE, H. (1950) Stress and the general adaptation syndrome. Br. reed. J. 1, 1387-1392. SH1NGAI, R., SUTHERLAND,M., ZAMAN, S., HARVEY, R., DARLISON, M. and BARNARD,E. A. (1990) Inhibitory effects of pregnenolone sulfate on cloned GABAA receptor subunits expressed in Xenopus oocytes. Soc. Neurosci. Abstr. 16, 380. SXMMONDS,M. A., TURNER,J. P. and HARRmON,N. L. (1984) Interactions of steroids with the GABA^ receptor complex. Neuropharmacology 23, 877-878. StaTS, S. S., WATEmtOUSE, B. D., CMAPIN, J. K. and WOOVW~D, D. J. (1987) Progesterone alters GABA and glutamate responsiveness: a possible mechanism of its anxiolytic action. Brain Res. 400, 353-359. TAYLOR,A., DAVIS,J. O. and JoI-r~SON,J. A. (1972) Control of deoxycorticosterone secretion in the dog. Am. J. Physiol. 223, 466-471. Ttmmm, D. M., RANSOM,R. W., YANO, J. S.-J. and OLSEN, R. W. (1989) Steroid anesthetics and naturally occurring analogs modulate the 7-aminobutyric acid receptor