View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLE published: 04 October 2011 provided by PubMed Central doi: 10.3389/fendo.2011.00044 Neurosteroids and GABA-A receptor function

Mingde Wang*

Section of Obstetrics and Gynecology, Department of Clinical Science, Umeå Neurosteroid Research Center, Umeå University, Umeå, Sweden

Edited by: Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, Hubert Vaudry, University of Rouen, the adrenals, and the gonads and have potent and selective effects on the GABAA- France receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, Reviewed by: Valerio Magnaghi, Università Degli and testosterone are positive modulators of GABAA-receptor in a non-genomic man- Studi di Milano, Italy ner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α,17α-diol (Adiol), and Giovanni Biggio, University of Cagliari, 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- cur- Italy rents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and Remy Schlichter, University α α α α Strasbourg – CNRS, France picrotoxin binding sites. 3 5 -P and 3 5 -THDOC potentiate synaptic GABAA-receptor δ *Correspondence: function and activate -subunit containing extrasynaptic receptors that mediate tonic Mingde Wang, Section of Obstetrics currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are and Gynecology, Department of GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. Clinical Science, Umeå Neurosteroid The activities of neurosteroid are dependent on brain regions and types of neurons. In Research Center, Umeå University, 901 85 Umeå, Sweden. addition to the slow genomic action of the parent steroids, the non-genomic, and rapid e-mail: [email protected] actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions.

Keywords: allopregnanolone, THDOC, pregnenolone sulfate, GABAA-receptor, premenstrual dysphoric disorder, mood, cognition

NEUROSTEROID in the CNS and alter neuronal excitability (Majewska et al., 1986; Sex hormones act through genomic mechanisms through the Paul and Purdy, 1992; Lambert et al., 1995). intracellular receptors located in the nucleus or cytoplasm. They The term “neurosteroid” was introduced to describe these act as ligand-activated transcription factors in the regulation of steroid metabolites that modulate neuronal activity (Paul gene expression. However, metabolites of progesterone and sev- and Purdy, 1992; Baulieu et al., 2007; Mellon, 2007). The eral stress hormones act on the membrane bound receptor via a 3α-hydroxy A-ring reduced metabolites of progesterone and non-genomic mechanism. The receptor binding to DNA and RNA deoxycorticosterone, allopregnanolone (3α5α-P), and 3α,5α- synthesis is thus not required (Frye et al., 1992; Baulieu and Robel, tetrahydrodeoxycorticosterone (3α5α-THDOC) were first shown 1995; Rupprecht, 2003). While the genomic action of sex hor- to modulate neuronal excitability by interaction with the GABAA- mones requires a time period from minutes to hours and limited receptor (Majewska et al., 1986). Several other neurotransmit- by the rate of protein biosynthesis (McEwen, 1991), the modula- ters like the NMDA, nicotinic, muscarinic, serotonergic, adren- tion on the membrane receptor is fast occurring event and requires ergic, and sigma 1 receptors are also targets for neurosteroids only milliseconds to seconds (McEwen, 1991). Today it is known (Klangkalya and Chan, 1988; Wu et al., 1991; Valera et al., 1992; that metabolites of sex and stress hormones act non-genomically Compagnone and Mellon, 2000; Mellon et al., 2001; Parry, 2001; Halbreich, 2003; Mellon, 2007). The functional modulation of the GABAA-receptor by neurosteroids at low concentrations is believed to induce moderate to severe adverse mood changes in Abbreviations: 3β5β-P, 3β-OH-5β-pregnan-20-one, epipregnanolone; 3β5α-P, 3β-OH-5α-pregnan-20-one, isoallopregnanolone; 3α5α-P, 3α-OH-5α-pregnan- up to 20% of female individuals (Beauchamp et al., 2000; Fish 20-one, allopregnanolone; 3α5β-P, 3α-OH-5β-pregnan-20-one, pregnanolone; et al., 2001). The clinical complex of premenstrual dystrophic 3α5α-THDOC, 3α,5α-tetrahydrodeoxycorticosterone; 3α5β-THDOC, 3α,5β- disorders (PMDD; Backstrom et al., 2003; Sundstrom Poromaa tetrahydrodeoxycorticosterone; 3β5β-THDOC, 5β-pregnan-3β, 21-diol-20-one; et al., 2003), petit mal epilepsy (Grunewald et al., 1992; Baner- α α α α α β β β 3 -adiol/3 5 -ADL, 5 -androstane-3 ,17-diol; 3 -HSD, 3 -hydroxysteroid jee and Snead, 1998), and catamenial epilepsy (Backstrom, 1976) dehydrogenase; 3α-OH, 3α-hydroxy; 3β-OH, 3β-hydroxy; CGC, cerebellar gran- ule cells; CNS, central nervous system; DGC, dentate gyrus granule cells; DHEAS, are among the disorders that may involve neurosteroid action. At dehydroepiandrosterone sulfate; DOC, deoxycorticosterone; EC50, concentration higher doses, neurosteroids may affect learning (Johansson et al., of drug that produces 50% of maximum response; GABA, γ-amino butyric acid; 2002), act as anxiolytic, anti-aggressive, sedative/anesthetic, and LTP, long-term potentiation; MPA, medroxyprogesterone; MPN, medial preoptic anti-epileptic agents in both animals and humans (Backstrom nucleus; nAChRs, nicotinic acetylcholine receptors; NMDA, N -methyl-d-aspartate; et al., 1990; Paul and Purdy, 1992; Wang et al., 2001; Bjorn et al., P45021, steroid 21 hydroxylase; P450c17, steroid 17 alpha-hydroxylase/17, 20 lyase; PMS/PMDD, premenstrual syndrome/premenstrual dysphoric disorder; PS, preg- 2002). nenolone sulfate; sIPSC,spontaneous inhibitory postsynaptic current; TBPS,t-butyl As shown in Figure 1, neurosteroids are synthesized in glial bicyclophosphorothionate; UC1011, 3β,20β-dihydroxy-5α-pregnane. cells and neurons of the central and peripheral nervous system

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from cholesterol or steroidal precursors imported from periph- progesterone and this conversion is mediated by P45021 (Edwards eral sources (Schumacher et al., 2000; Baulieu et al., 2007). They et al., 2005). The conversion of DOC to 3α5α-THDOC occurs include 3β-hydroxy-Δ5-compounds such as pregnenolone and both in peripheral tissues and in the brain (Reddy, 2003). The dehydroepiandrosterone, their sulfate esters and reduced metabo- A-ring reduced metabolite of testosterone, 5α-androstane-3α,17β- lites of steroid and stress hormones such as the tetrahydroderiv- diol (3α5α-adiol), acts also as a GABAA-receptor agonist (Frye ative of progesterone, 3α-hydroxy-5α-pregnan-20-one (allopreg- et al., 1996). nanolone; Baulieu et al., 2007). Progesterone itself is also a neuros- teroid, and a progesterone receptor has been detected in peripheral GABAA-RECEPTOR and central glial cells (Schumacher et al.,2000; Baulieu et al.,2007). GABA mediates most of the inhibitory neurotransmission in the At different sites in the brain, concentrations of neurosteroids vary mammalian brain. GABA-mediated inhibition is crucially in both according to environmental and behavioral circumstances, such as short-term and long-term regulation of neuronal excitability. It stress, sex recognition, and aggressiveness. Allopregnanolone can has been estimated that approximately 33% of the synapses in the accumulate in the brain after adrenalectomy and gonadectomy mammalian cerebral cortex are GABAergic (Purvez et al., 2004). (Purdy et al., 1991; Corpechot et al., 1993; Cheney et al., 1995). Two major types of receptors, GABAA- and GABAB-receptors can This indicates that allopregnanolone is synthesized in the brain be identified in the CNS (Sivilotti and Nistri, 1991; Bormann, via A-ring reduction of progesterone (Celotti et al., 1992; Do Rego 2000). It appears that neurosteroids more or less exclusively tar- et al., 2009). Table 1 shows concentrations of major neurosteroids get the GABAA-receptor which is a ligand gated anion-selective in the plasma and brain. channel (Schofield et al., 1987). Pregnenolone sulfate (PS) and dehydroepiandrosterone sul- Various isoforms of the GABAA-receptorhavebeenidentified fate (DHEAS) are naturally occurring neurosteroids that inhibit that comprise α1–6, β1–3, γ1–3, δ, ε, π, θ, and ρ1–3 subunits (Amin the GABAA-receptor (Paul and Purdy, 1992). PS is synthesized and Weiss, 1994; Mehta and Ticku, 1999; Rudolph et al., 2001). In from pregnenolone by the enzyme sulfotransferase (Figure 1). general, GABAA-receptors are pentameric proteins (Nayeem et al., Conversion from DHEA to DHEAS is also mediated by sulfotrans- 1994) that are built of five subunits which includes two α-subunits, ferase. DHEA is metabolized from pregnenolone by cytochrome two β-subunits and one subunit of either the γ-,δ-,ε-,π-,or θ-type P450C17 (Mensah-Nyagan et al., 1999). On the other hand, 3β- (Farrar et al., 1999; Knight et al., 2000; Klausberger et al., 2001). HSD is essential for the synthesis of 3β-OH steroids, i.e., 3β-OH- A subgroup of GABAA-receptor channels was named as GABAC- 5α-pregnan-20-one (isoallopregnanolone; 3β5α-P) and 3β-OH- receptor earlier and composed of homo-oligomeric ρ1–3 subunits. 5β-pregnan-20-one (epipregnanolone; 3β5β-P; Stromstedt et al., They are pharmacologically distinct from other GABAA-receptor 1993). PS, DHEAS, and 3β-OH steroids act as antagonists on the channels and this difference is illustrated by the insensitivity of ρ GABAA-receptor (Wang et al., 2002; Eisenman et al., 2003) and receptor channels to many known modulators such as barbiturates can be measured from human blood samples (Hill et al., 2001). and benzodiazepine (Amin and Weiss, 1994, 1996). As metabolites of stress hormone deoxycorticosterone (DOC), Immunological, pharmacological, and functional analysis give 3α5α-THDOC and 3α5β-THDOC are also potent modulators of the evidence that the α1β2γ2 combination is the most common the GABAA-receptor (Crawley et al., 1986; Majewska et al., 1986; GABAA-receptor within the CNS (∼60%), followed by α2β3γ2 Gasior et al., 1999; Lambert et al., 2001a). Both steroids have sig- (∼15–20%) and α3βnγ2 (∼10–15%, n = 1, 2, or 3; Mohler et al., nificant sedative effects in vivo.3α5α-THDOC is responsible for 2002, 2004; Fritschy and Brunig, 2003; Wallner et al., 2003). Recep- the sedative and anti-seizure activity of DOC in animal mod- tors containing the α4-, α5-, and α6-subnit, as well as the β1-, γ1–3, els (Reddy and Rogawski, 2002). DOC can be metabolized from δ-, π-, and θ-subunit, form a minor receptor population. Each of

Table 1 | Neurosteroid concentrations in human plasma and brain (mean ± SEM).

Steroids Plasma (nM) Brain (nM)

Follicular Luteal Postmenopausal Luteal

Progesterone 5.0 ± 0.50 (Wang et al., 1996) 34.7 ± 2.40 65 (Bixo et al., 1997) 137 (Bixo et al., 1997) 3α-OH-5β-pregnan-20-one 0.6 ± 0.00 (Sundstrom et al., 1998)1.1± 0.50 – 114 (Bixo et al., 1997) 3α-OH-5α-pregnan-20-one 0.2–0.6 (Wang et al., 1996; 2–4 (Wang et al., 1996; 47 (Bixo et al., 1997)66(Bixo et al., 1997) Genazzani et al., 1998) Genazzani et al., 1998) 3β-OH-5α-pregnan-20-one 0.3–0.5 (Wang et al., 1996; 1.5–3.5 (Wang et al., 1996; –– Sundstrom et al., 1998)) Sundstrom et al., 1998) 3β-OH-5β-pregnan-20-one 0.09 ± 0.08 (Havlikova et al., 2006) 0.26 ± 0.13 – – Pregnenolone sulfate 11.2 ± 0.6 (Wang et al., 1996) 15.2 ± 0.8 (Bixo et al., 1997)– 100(Lanthier and Pat- wardhan, 1986) Pregnenolone *2.19 (Kancheva et al., 2007)– – – 3α5α-androstane-3α,17β-diol *0.475 (Kancheva et al., 2007)– – –

Data cited from references are inserted as indicated. *Concentration in adult men.

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FIGURE 1 | Biosynthesis of allopregnanolone and pregnenolone 5α-reductase. Transport of cholesterol across the mitochondrial membrane sulfate (PS) from cholesterol within the neuron or glial cell. Enzymes is enhanced by the steroidogenic acute-regulatory (StAR) protein and the involved are P450 side-chain cleavage (P450scc), 3α-hydroxysteroid mitochondrial benzodiazepine receptor (MBR). 5α-DHP represents dehydrogenase (3α-HSD), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 5α-dihydroprogesterone.

the α4βnδ, α4βnγ, and the α6β2/3γ2 receptor accounts less than 5% it is localized to interneurons in the hippocampus and cortex of all the GABAA-receptor quantity (McKernan andWhiting,1996; (layer I–IV), and cerebral Purkinje cells (McKernan and Whiting, Whiting, 2003a,b). The α6βnδ receptor has a small population in 1996). The α2β3γ2 receptor is present in cerebral cortex (layer I– the cerebellum and the α6β2/3γ2 receptor located exclusively in the IV), hippocampal formation, amygdale, striatum, olfactory bulb, cerebellum (McKernan and Whiting, 1996; Whiting, 2003a,b). hypothalamus, superior colliculi, and motor nuclei (Fritschy and The expression of GABAA-receptor subtypes in the adult brain Brunig, 2003). The α3βnγ2, α3γ2, and α3θ receptors are abun- exhibits a remarkable regional and neuronal specificity which sug- dant in the cerebral cortex (layers V–VI), amygdala, olfactory bulb, gests that individual subtypes are present in distinct neuronal thalamic reticular and intralaminar nuclei, superior colliculus, circuits. The α1β2γ2 receptor is present in most brain areas and brainstem, spinal cord, and locus coeruleus. The α4βnδ (n = 1, 2,

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or 3) receptor is presented in the dentate gyrus and thalamus. The the concept that GABAA-receptor heterogeneity is a major facet α5β3γ2 receptor is widespread in the hippocampus and dentate determining the functional properties of GABAergic inhibitory gyrus (Glykys et al., 2008), deep cortical layers, amygdala, olfactory circuits (Sieghart, 2000; Mohler et al., 2001). In particular, the type bulb, hypothalamus, superior colliculus, superior olivary nucleus, of α-subunit determines the kinetics of receptor deactivation (Ver- spinal trigeminal nucleus, and spinal cord. The α6β2/3γ2, α6β2/3δ, doorn et al., 1990; Hutcheon et al., 2000; Devor et al., 2001), and and α6β2/3γ2 receptors are found mainly in the cerebellum and the presence of the δ-subunit results in markedly increased agonist dorsal cochlear nucleus (Fritschy and Brunig, 2003). affinity and apparent lack of desensitization (Burgard et al., 1996; Fisher and Macdonald, 1997; Adkins et al., 2001). PHARMACOLOGICAL RELEVANCE OF GABAA-RECEPTOR SUBUNIT The GABAA-receptor expresses different subunit compositions COMPOSITIONS in different parts of the brain (Sieghart and Sperk, 2002). The Especially the different α-subunits have been attributed to spe- subunit composition is related to different function of the spe- cific behavioral effects as shown in Table 2. For example, an cific part of the brain (Korpi et al., 2007; Table 2). This anatomical enhancement at the α1 subunit has been associated with seda- diversity constitutes the very basis for the pathogenesis of different tion (Rudolph et al., 1999) and the α2 with anxiolytic action. conditions. Steroids interact differently with the GABAA-receptor Recently, it has been shown that the α5 subunit is important depending on the subunit composition (Belelli et al., 2002). The for sedative tolerance development to benzodiazepines and for α5 subunit is localized in high degree in the hippocampus, a key acquisition and expression of associative memory and spatial area for memory and learning, α5 is shown to be important for learning (Collinson et al., 2002; Crestani et al., 2002; van Rijn- learning and memory function (Table 2) since α5-subunit knock- soever et al., 2004; Yee et al., 2004). In addition, the α4 subunit out mice in comparison with wild-type mice show significantly is also implicated in the regulation of anxiety (Gulinello et al., better performance in a water maze model of spatial learning 2001). A concentration-dependent decrease of the α4 subunit is (Collinson et al., 2002). In addition, blockade of the GABAA- seen after 4-day application of allopregnanolone to developing receptor subunit α5 increased learning and memory (Casula et al., neuronal cells (Grobin and Morrow, 2000). In the hippocampus 2001; Maubach,2003). GABAA-receptor activation can inhibit LTP and cerebellum, an increase of the α4 subunit can be detected after induction and NMDA receptors, which are involved in the regu- withdrawal from chronic exposure and after short-term treatment lation of hippocampal-dependent spatial memory (Riedel et al., of progesterone and allopregnanolone (Smith et al., 1998; Concas 2003). et al., 1999; Follesa et al., 2001; Gulinello et al., 2001). The α6 sub- Several papers report changes in the GABAA-receptor sub- unit is highly sensitive to pentobarbital (Thompson et al., 1996) unit composition and decreased GABA function after long-term and neurosteroids (Belelli et al., 2002). exposure to GABAA-receptor agonists (Miller et al., 1988; Belelli The γ2 subunit is also involved in anxiety regulation, and it et al., 2002). It is well-known that tolerance develops during long- is changed during hormone treatment and pregnancy (Essrich term GABAA-receptor exposure. Tolerance development is noted et al., 1998; Follesa et al., 1998; Concas et al., 1999; Crestani already after 90 min exposure to anesthetic dosages of allopreg- et al., 1999; Kittler et al., 2000). The δ-subunit is responsible nanolone. Changes in the α4 subunit of the GABAA-receptor in for tonic conductance and important for neurosteroid modula- thalamus were related to the tolerance development (Birzniece tion on GABAA-receptor (Stell et al., 2003). Interestingly, receptor et al., 2006a). GABA-steroids are positive neurosteroid modula- knockout studies have revealed that the absence of the δ-subunit tors on the GABAA-receptor (Majewska et al., 1986). Long-term decreases the sensitivity to neurosteroids such as pregnanolone treatment with GABA-steroids may induce down-regulation of the and alphaxalone, thereby influencing the duration of anesthesia GABAA-receptor function in mammalian cultured neurons both and the anxiolytic effect of those steroids (Mihalek et al., 1999). for neuroactive steroids and other GABAA-receptor active drugs Finally, the ε-subunit reduces neurosteroid and anesthetic mod- (Yu and Ticku, 1995a; Yu et al., 1996). During pregnancy when ulation (Davies et al., 1997; Belelli et al., 2002; Thompson et al., allopregnanolone is high there is a decrease in GABAA-receptor 2002). function and changes in subunit composition of the GABAA- Functionally, distinct subunit-specific properties have been receptor. Inhibiting the synthesis of allopregnanolone blocked identified in both recombinant and native receptors, supporting these changes (Concas et al., 1998). Chronically high cortisol levels

Table 2 | Relationship between behavior disorders/symptoms, brain areas, and subunits of the GABAA-receptor.

Behavior disorders/symptoms Brain region GABAA-receptor subunit

Memory and learning disruption Hippocampus α5 δ

Anxiety and mood disturbance Hippocampus; amygdale α2 α4 β3 δ

Fatigue, sedation, and exhaustion Wide spread α1

Depression induced by stress α1 α3 β1 β2 δγ2

Eating disorders Hypothalamus α3

Relapse in alcohol abuse β6

Balance and mobility disorders Cerebellum α6 β2 δ

Epilepsy and excitability disorders α1 β3

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and GABA-steroids give irreversible cognitive damages. A reduced located opposite to the release site (Mody et al., 1994; Nusser et al., sensitivity to benzodiazepines, alcohol, and GABA-steroid is also 1997; Brickley et al., 1999). seen in women with PMDD during the luteal phase. Such a change Synaptically released GABA which act on postsynaptic GABAA- in GABAA-receptor sensitivity, as measured by reduced sedation receptors is termed “phasic” inhibition, whereas the term “tonic” and saccadic eye velocity response to GABA active compounds, inhibition refers to a continuous activation of extrasynaptic recep- contributes to symptom severity of PMDD patients (Backstrom tors by ambient GABA (Farrant and Nusser, 2005). The main et al., 2003). In a rat model of PMDD, allopregnanolone upreg- feature of phasic inhibition is the rapid synchronous opening of a ulates the α4 subunit of the GABAA-receptor in hippocampus relatively small number of channels that are clustered within the parallel to the induction of anxiety (Smith et al., 1998; Gulinello synaptic cleft. This enables a resolution both in time (the release et al., 2001). In addition, the anxiety induction was blocked if is triggered by an incoming action potential) as well as in space the animals were treated with α4-antisense (Smith et al., 1998; (the release is limited and thereby the postsynaptic action, to a Gulinello et al., 2001). With a further developed rat model of specific synapse. In contrast, tonic inhibition results from ambi- PMDD, the authors addressed that the high risk-taking rats are ent GABA and therefore is relatively constant in both time and those react with anxiety in the PMDD model (Lofgren et al., 2006). space. Although both modes of action clearly impact upon neu- ronal information processing at the cellular and network level, the SYNAPTIC AND EXTRASYNAPTIC GABAA-RECEPTORS extent to which each type of inhibition influences brain excitabil- The GABAA-receptors can either be synaptic (located within ity in normal and diseased states is not known. GABA-mediated the synaptic cleft) or extrasynaptic (located outside the synaptic tonic conductance is found in granule cells of the dentate gyrus cleft; Fortin et al., 2004). The synaptic receptors usually contain (Nusser and Mody, 2002; Farrant and Nusser, 2005), thalamocor- γ-subunits and can be rapidly expressed at the neuronal mem- tical relay neurons of the ventral basal complex (Porcello et al., brane and are sensitive to both benzodiazepines and neurosteroids. 2003), layer V pyramidal neurons in the somatosensory cortex Extrasynaptic GABAA-receptors are in a preferred position to be (Yamada et al., 2004), CA1 pyramidal cells (Bai et al., 2001), and activated by the low levels of ambient GABA, due to their high certain inhibitory interneurons in the CA1 region of the hip- GABA affinity in contrast to the lower affinity of synaptic GABAA - pocampus (Semyanov et al., 2003). Unlike receptors mediating receptors (Saxena and Macdonald, 1994; Mody, 2001; Brown et al., phasic current, tonically active GABAA-receptors show unusual 2002; Farrant and Nusser, 2005). The high-affinity extrasynaptic high GABA affinity (Saxena and Macdonald, 1996) and be acti- GABAA-receptors consist of specific subunit combinations dif- vated by the low ambient GABA concentrations (nanomolar to ferentially expressed in various brain regions. These include the few micromolar; Lerma et al., 1986; Tossman et al., 1986). At δ-subunit containing GABAA-receptors of dentate gyrus and cere- sub-micromolar concentrations, several competitive, and non- bellar granule cells, cortical and thala mic neurons (Nusser et al., competitive GABAA-receptor antagonists (gabazine, picrotoxin, 1998; Sur et al., 1999; Pirker et al., 2000; Nusser and Mody, 2002; and bicuculline) reduced phasic currents, but had no effect on Stell et al., 2003; Sun et al., 2004; Cope et al., 2005; Jia et al., tonic currents. However, the antagonists blocked both phasic and 2005; Drasbek and Jensen, 2006), and the α5-subunit containing tonic currents at high concentrations (Bai et al., 2001; Stell and GABAA-receptors in CA1 and CA3 pyramidal cells (Sperk et al., Mody, 2002; Semyanov et al., 2003; Yeung et al., 2003; Farrant and 1997; Caraiscos et al., 2004; Glykys and Mody, 2006). The cur- Nusser, 2005). rent mediated by these extrasynaptic receptors has been termed tonic inhibition (Brickley et al., 1996; Farrant and Nusser, 2005), THE EFFECT OF NEUROSTEROIDS ON THE GABAA-RECEPTOR which is highly sensitive to the extracellular GABA concentra- The GABAA-receptor can be modulated by a number of therapeu- tion (GABA). It is enhanced when ambient (GABA) is increased tic agents, including benzodiazepines (Sieghart, 1992; Macdonald by blocking GABA transporters, by adding GABA to the aCSF and Olsen, 1994), barbiturates (Smith and Riskin, 1991), anes- to mimic that normally present in the extracellular space, or by thetics, ethanol (Harris et al., 1995), zinc (Smart, 1992), and preventing GABA degradation (Nusser and Mody, 2002; Stell and neurosteroids (Puia et al., 1990; Hawkinson et al., 1994a; Lam- Mody, 2002; Wu et al., 2003; Glykys and Mody, 2006). bert et al., 2001b). The effect of neurosteroids on the GABAA- The δ-subunit is highly sensitive to GABA and neurosteroids receptor depends on the type of steroids (agonist or antagonist), but not to benzodiazepines (Brown et al., 2002; Smith et al., 2007). the type of receptors (synaptic of extrasynaptic), the subunit It also appears that specific types of receptor subunit combina- compositions, and the intrinsic structure of the steroid. Recent tions are expressed in the synaptic cleft and extra synaptically. For studies indicate that the existence of at least two neurosteroid example, the α1β2/3γ2, α2β2/3γ2, and α3β2/3γ2 receptors are the actions on the GABAA-receptor, namely an agonistic action and predominant synaptic receptors (Farrant and Nusser, 2005) and an antagonistic action by the sulfated and 3β-OH steroids. The the combinations of α6βnδ, α4βnδ, α1βnδ, and α5βnγ2 receptors, agonistic action can further be divided into an allosteric enhance- are predominantly or exclusively extrasynaptic (Nusser et al., 1998; ment of GABA-evoked Cl− current and a direct activation of the Fritschy and Brunig,2003; Glykys and Mody,2007). In the synapse, GABAA-receptor. each vesicle is thought to release several thousands of GABA mol- The synaptic currents originates from a short exposure of ecules into the synaptic cleft which leads to a high concentration high concentrations of GABA that induces receptor desensitiza- of GABA (0.3–1.0 mM) in a time span of 10–100 ms (Mozrzymas tion (Jones and Westbrook, 1995). As a consequence of this, the et al., 2003; Semyanov et al., 2004). Furthermore, it is suggested mechanism of neurosteroid modulation at the GABAA-receptor that there are rather few receptors, from 10 to a few hundreds, cannot easily be predicted by studies of currents evoked by low

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concentrations of GABA. It has been shown that allopregnanolone sensitivity compare to γ-subunit containing receptor (Davies et al., remarkably increases the decay time of sIPSC (Haage and Johans- 1997; Belelli et al., 2002). Receptors incorporating the ε-subunit son, 1999; Belelli and Lambert, 2005), which likely depends on a are reported to be insensitive to the modulation by pregnane reduced GABA unbinding rate from the receptor. However, a dif- steroids, not the direct GABA-mimetic effect (Lambert et al., ferent mechanism was suggested for the 3α5α-THDOC induced 2001b). decay time prolongation. Here it was instead suggested that the underlying mechanism was altered kinetics of desensitized states SYNAPTIC EFFECT OF NEUROSTEROIDS (Zhu and Vicini, 1997). The above explanations for the mecha- The brief inhibitory response of neurosteroids by activating nisms underlying the effects of neurosteroids is based on the idea the postsynaptic GABAA-receptor is a phasic response. Synaptic that once GABA is bound to the receptor it can enter different GABAA-receptors are ternary complexes that commonly incorpo- states of which some are open and some are closed (desensitized) rate the γ2 subunit in combination with one α (mainly α1/2/3) and which will determine the shape of the evoked current (Jones and one β2/3 subunit. However, these receptor isoforms can also be Westbrook, 1995). located extrasynaptically (Farrant and Nusser, 2005). The kinetic At low-micromolar concentrations of GABA,allopregnanolone of agonist steroids at synaptic GABAA-receptor has been stud- instead increases the activation rate and induces a more prominent ied thoroughly by measuring the sIPSC from neurons in brain desensitization of GABA-evoked currents (Haage and Johans- slice. Neurosteroids have little effect on the onset time and peak son, 1999). Interestingly, there is also evidence that the effect amplitude of the sIPSC. Agonist neurosteroids prolong the decay of 3α5α-THDOC to prolong the deactivation time (which is the time constant of IPSC (Majewska et al., 1986; Zhu and Vicini, same as the decay time but is used for externally applied GABA) 1997; Haage et al., 2005). However, this effect is neuron specific. In depends on the age. The effect on the deactivation of GABA hippocampal CA1 neurons, cerebellar granule cells and Purkinje response in cerebellar neurons is greater in younger rats than neurons, neurosteroids prolong the sIPSC at relatively low concen- adult rats (Zhu and Vicini, 1997). In summary, we believe that tration (in the nanomolar range; Cooper et al., 1999; Vicini et al., the prolongation of deactivation and altered receptor kinetics 2002; Harney et al.,2003). On the other hand, micromolar concen- in terms of entry and exit from desensitized states, are essential trations are required to produce equivalent responses in oxytocin to the allosteric modulation of the GABAA-receptor by neuros- neurons of hypothalamus (Brussaard et al., 1997; Koksma et al., teroids. Agonistic neurosteroids affect not only the time course 2003). Moreover,in the preoptic cells in the hypothalamus,100 nM of sIPSC but also the frequency of the sIPSCs. Such an increase allopregnanolone prolong the spontaneous current (Haage et al., in frequency is due to the presynaptic effect of neurosteroids 2005; Stromberg et al., 2006). This indicates that the neurons in (Poisbeau et al., 1997; Haage et al., 2002) by a mechanism that the same brain region can show heterogeneity. In addition, the involves altered presynaptic Ca2+ permeability and activation of effect of 3α5α-THDOC on GABA-binding kinetic is more pro- presynaptic GABAA-receptors. found in the hippocampal CA3 and subiculum than that in CA1 The effect of neurosteroids on the GABAA-receptor can be and entorhinal cortex (Nguyen et al., 1995). At higher concen- attributable to variations in the receptor subunit composition. trations (>10 μM) which can occur in the brain during partu- The action of allopregnanolone was not influenced by the α- rition (Stoffel-Wagner, 2003), neurosteroids activate the GABAA- subunit, when co-expressed with β1- and γ2-subunit in Xenopus receptor directly (Majewska et al., 1986) in a similar pattern as oocytes (Belelli et al., 2006). The GABA responses at α1β1γ2 and barbiturates by interacting with different sites on GABAA-receptor α3β1γ2 receptor are enhanced by low concentration of allopreg- (Kerr and Ong, 1992). This “GABA-mimetic” effect of neuros- nanolone (≥3 nM), whereas several folds higher concentrations teroid is sufficient to suppress the excitatory neurotransmission are needed to obtain the equivalent response on α2-,α4-, α5-, (Shu et al., 2004). or α6-subunit containing receptors. Likewise, the subtypes of the β-subunit (β1–3) have little action on the effects of neu- EXTRASYNAPTIC EFFECT OF NEUROSTEROIDS rosteroids (Hadingham et al., 1993; Sanna et al., 1997; Belelli The response of neurosteroids at relatively low concentrations et al., 2002). The presence of a γ-subunit is not a prerequisite is mediated by the activation of extrasynaptic GABAA-receptors for the neurosteroid activity. In fact, the efficacy of allopreg- containing α4, α6, and δ-subunits. Extrasynaptic receptors iden- nanolone action at the binary α1β1 receptor is higher than that tified at the granule cells of the dentate gyrus and cerebellum, at the ternary α1β1γ2 receptors (Maitra and Reynolds, 1999; and the relay neurons of the thalamus, are distinct from the Belelli et al., 2002). Given the γ-subunit have little or no effect synaptic receptors. Extrasynaptic conductance can have a con- on the maximal GABA-modulation effect of allopregnanolone, it siderable influence on neuronal excitability (Leroy et al., 2004). significantly influences the potency of the steroid with “physio- Extrasynaptic receptors exhibit both a high GABA affinity and logical concentrations” (3–30 nM; Belelli et al., 2002). However, reduced receptor desensitization in the continued presence of the inhibition of GABAA-receptor by PS did not vary between the agonist (Fritschy and Brunig, 2003). Such properties ren- binary and ternary receptor (Wang et al., 2006). The poten- der these receptors ideally suited to sense the low ambient con- cies and efficacies of PS to inhibit GABA saturating concen- centrations (∼0.5–1 μM) of the extrasynaptic neurotransmitters tration at the α1β2γ2L and α1β2 receptor were identical (Wang (Kennedy et al., 2002). Extrasynaptic receptors containing the et al., 2006). On the other hand, δ-subunit when co-expressed δ-subunit are highly sensitive to neurosteroids in certain brain with α4- and β3-subunits, a receptor thought to be naturally region (Wohlfarth et al., 2002). At low “physiological” concentra- present in the thalamus (Sur et al., 1999) shows high steroid tions (10–100 nM), 3α5α-THDOC selectively enhance the tonic

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conductance, with little or no effect on the phasic conductance in mouse DGCs and CGCs (Stell et al., 2003; Belelli and Lam- bert, 2005; Farrant and Nusser, 2005). Tonic inhibition is reduced in the δ-subunit “knockout” mice, and the residual tonic cur- rent was insensitive to 3α5α-THDOC (Mihalek et al., 1999; Stell et al., 2003). However, the extrasynaptic effects of neurosteroids also reveal regional difference in the CNS. Two hundred fifty nanomolar 3α5α-THDOC has no effect on the tonic inhibition in ventrobasalis complex of the thalamus (Porcello et al., 2003). On the other hand, the tonic conductance of hippocampal CA1 neurons expressing GABAA-receptor with α5 subunit is affected by 3α5α-THDOC (≥100 nM; Stell et al., 2003; Belelli and Lam- bert, 2005; Farrant and Nusser, 2005). It is suggested that the FIGURE2|Featuresofgeneral structure of the neurosteroid. Figure shows intact four ring system that often contains hydroxyl side chains as modulation of tonic currents is influenced by local neurosteroid sterols. Hydroxyl groups are denoted α if they are oriented above the plane metabolism. The inhibition of metabolism can greatly enhance (solid line), and α if they are oriented below the plane (dashed line). the response of the tonic current in dentate granule cells to Stereoisomerism and structural modifications were discussed at carbon 3, endogenous neurosteroids, but not on the synthetic metaboli- 5, 6, 7, 17, 20, and 21 positions. cally stable ganaxalone (Belelli and Herd, 2003). Neurosteroid metabolism reveals regional specificity that also contributes to 7-methanoindene derivatives give a compound that showed weak the regional specificity of the tonic GABAA-receptor mediated potentiating activity (Burden et al., 2000). When the carbonitrile currents. In summary, evidence is emerging that the GABAA- receptor mediated tonic conductance present in some neurons is replaced with an acetyl group the derivatives become inactive may have a considerable influence on neuronal signaling and (Hamilton, 2002). network activity (Brickley et al., 2001; Hamann et al., 2002; The configuration of C5-reduction is important to potency. α β Mitchell and Silver, 2003). The high sensitivity of neurosteroids Even if steroids with either 5 or 5 conformations are active, spa- in extrasynaptic receptor may represent an important target for tial difference in this position may affect the pharmacology of the α neurosteroids. neurosteroids. Allopregnanolone with 5 -reduction is generally more potent than its 5β-isomer,pregnanolone,as GABAA-receptor α α AGONIST NEUROSTEROIDS ON THE GABA -RECEPTOR agonist both in vivo and in vitro. Studies with 3 5 -THDOC and A α β The structure–activity relationship of neurosteroid actions on the its stereoisomer 3 5 -THDOC revealed important differences in GABA -receptor has been summarized in a number of articles potency, efficacy, and regional selectivity at the GABAA-receptor in A α (Laubach et al., 1955; Gyermek et al., 1968; Gyermek and Soyka, favor of 5 -reduction (Gee and Lan, 1991; Mennerick et al., 2004). α β 1975; Sear, 1997). The systemic investigation of several isomers 5 -steroid, but not 5 -steroids, showed a high degree of enantios- electivity/enantiospecificity in their action as modulator of the of GABAA-receptor active neurosteroids revealed a couple crucial structure variations (Figure 2), namely the geometry between ring GABAA-receptor and as anesthetics (Covey et al., 2000). The effi- β β A/B; a hydrogen-bond donator in C3 position; a hydrogen-bond cacy of 3 5 -P to inhibit GABAA-receptor is significantly different β α β acceptor in C20 position and/or a flexible bond at C17 position from 3 5 -P (Rahman et al., 2006; Wang et al., 2007). 5 -reduced (Purdy et al., 1990; Zorumski et al., 2000; Ragagnin et al., 2007). antagonist neurosteroids cause significant higher inhibition than α The α- and β-configuration refer to constituents below and above the 5 -isomers. the plane of the steroid backbone. Variation of the core and sub- Like pregnan steroids, metabolites of testosterone such as stitution in certain positions of the steroid molecules has crucial androstanediol are also modulators of GABA. It was found that α consequence on the neurosteroid effects (Figure 2). the systemic 3 -androstanediol administration conditions a place Anesthetic steroids typically have a saturated backbone of four preference more effectively than does systemic administration rings, although this is not an absolute requirement for activity. of dihydrotestosterone or testosterone (Frye, 2001). Additionally, α The four rings form a rigid framework for positioning the hydro- plasma concentration of 3 -androstanediol is increased compare gen bonding groups in three-dimensional spaces (Figure 2). The to dihydrotestosterone and testosterone. However, the potency α presence of hydrogen-bond donor in the α-configuration at C3 and efficacy effect of 3 -androstane steroid is lower than those α and β-configuration at C17 are critical for the sedative action of of 3 -pregnan steroids (Rahman et al., 2006). agonist neurosteroids (Purdy et al., 1990; Hawkinson et al., 1994b; Hogenkamp et al., 1997). These groups are important for the bind- ANTAGONIST NEUROSTEROIDS ON THE GABAA-RECEPTOR ing of neurosteroids to a variety of proteins by means of hydrogen- Neurosteroids may both enhance and inhibit GABAergic neuro- binding with polar or charged residues (Brzozowski et al., 1997; transmission (Wang et al., 2002; Mennerick et al., 2004; Rahman Grishkovskaya et al., 2000). Replacing the hydrogen of hydroxyl et al., 2006; Stromberg et al., 2006). It has been shown earlier that with methyl, thus eliminating the ability of the steroid to donate a 3β-hydroxy A-ring reduced pregnane steroids (3β-OH steroids) hydrogen bond in this region, results in dramatic reduction in and pregnenolone sulfate inhibit GABAA-receptor coupled anion its potency (Upasani et al., 1997). Replacing the steroid skele- channels (Wang et al., 2002, 2007; Lundgren et al., 2003; Birzniece ton with an alicyclic framework like 2-cyclohexylideneperhydro-4, et al., 2006b). 3β-OH steroids and PS, at concentrations that had

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little effect on GABAergic synaptic currents, significantly reversed leave open the possibility that PS may prefer liganded over unli- the potentiating effect of 3α-OH A-ring reduced steroids (Wang ganded receptors, consistent with a model of state-dependence to et al., 2002). Although antagonistic steroids reduced the potentia- PS actions. tion induced by 3α-OH steroids, they still acted non-competitively An earlier study observed that PS inhibits GABA-gated Cl− cur- with respect to the agonistic steroids and inhibited a larger poten- rent by enhancing receptor desensitization and stabilizing desen- tiation more efficiently (Wang et al., 2002). Furthermore, 3β-OH sitized state with prolonged application of low-affinity GABA steroids co-applied with GABA alone significantly inhibited GABA agonists to nucleated membrane patched (Shen et al., 2000). Note responses at concentrations ≥EC50 (Garrett and Gan,1998; Maitra that the promotion of desensitization is selective for prolonged and Reynolds, 1998; Wang et al., 2002; Lundgren et al., 2003). This GABA applications (Shen et al., 2000). It is well-known that the direct, non-competitive effect of 3β-OH steroids and PS on the time course of GABA-mediated IPSCs is influenced strongly by GABA response was sufficient to account for the apparent antago- the kinetics of the GABAA-receptor desensitization (Celentano nism of agonist steroids (Rahman et al., 2006). In summary, both and Wong, 1994; Jones and Westbrook, 1995). Desensitized states PS and 3β-OH steroids inhibited the GABAA-receptor more effec- are thought to buffer receptors in bound conformations that tively under conditions that promoted agonist binding or channel make it possible for channels to re-open before GABA unbinds. opening (Wang et al., 2002). The interaction between antagonist The fast phase of desensitization limits the open probability of and agonist steroids was due to a use-dependent action of antago- the channels, influences peak synaptic currents, and contributes nist steroids at recombinant and synaptic GABAA-receptors (Wang to the fast component of IPSC decay. The slow component of et al., 2002). decay may result from reopening of GABA channels after exit from desensitized states (Jones and Westbrook, 1995). On the MECHANISM OF PREGNENOLONE SULFATE AND NEUROSTEROID other hand, recombinant GABAA-receptors composed of defined SULFATE ESTERS subunit combinations also give rise to currents with complex Sulfated steroids like pregnenolone sulfate and dehydroepiandros- decay kinetics (Lavoie et al., 1997; Haas and Macdonald, 1999). terone sulfate can produce profound effects on behavior. PS as In cultured hippocampal neurons, PS decreases the peak current an abundant neurosteroid enhances learning (Mayo et al., 1993; of the inhibitory autapic currents, enhances the fast and slow Flood et al., 1995), and antagonizes the impairment of learning phases of deactivation after brief application of a few millisec- and memory produced by ethanol and scopolamine (Melchior and onds. After longer application (∼100 ms or more), PS enhances Ritzmann, 1996). PS may play a role in cognition and have been both the fast and slow phases of desensitization of the GABA reported as negative modulators of the GABAA-receptor based on current (Shen et al., 2000; Eisenman et al., 2003). Moreover, electrophysiological studies and GABA-mediated 36Cl− uptake by PS reduces the agonist steroid evoked prolongation of sIPSC rat brain synaptosomes (Majewska et al., 1990; Demirgoren et al., and reduces channel-opening frequency (Mienville and Vicini, 1991). Structure–activity relationships of GABAA-receptor modu- 1989). However, recording the sIPSC of neurons medial preoptic lation are different for sulfated inhibitory steroids vs. non-sulfated nucleus (MPN) shows that PS neither affects the peak ampli- potentiating steroids (Park-Chung et al., 1999). Potentiation by tude nor the decay of sIPSC (Haage et al., 2005). Instead, PS non-sulfated steroids requires 3α-stereochemistry. Pregnenolone reduces the frequency of the sIPSC at higher concentration (Haage by itself is inactive on the GABAA-receptor. In contrast, both 3α- et al., 2005). The main effect of PS on the sIPSC time course is and 3β-isomers of PS is inhibitory. Although the addition of a explained by a simplified model that this substance reduce the rate negatively charged sulfate group or hemisuccinate group at the of desensitization while the agonist steroids assumed to reduce C3 position converts the neurosteroid from being potentiating to the unbinding rate of GABA from the receptor (Haage et al., inhibitory (Park-Chung et al., 1999), a negatively charged group 2005). at C3 is not absolutely essential for inhibition since the non- sulfated neurosteroid DHEA is also inhibitory. However, DHEA MECHANISM OF 3β-HYDROXYSTEROID AS GABAA-RECEPTOR is less potent than its sulfated derivative DHEAS (Imamura and ANTAGONIST Prasad, 1998; Park-Chung et al., 1999). Steroids such as 11-keto According to earlier studies, PS (Woodward et al., 1992), 3β- derivative of PS are of particular interest, as it behaves as a posi- OH steroids, or carboxylated steroids (Mennerick et al., 2001) tive, negative, or neutral modulator on the GABAA-receptor. It is are more effective against GABA responses gated by high con- suggested that 11-keto pregnenolone sulfate (11-keto PS) exerts centrations of GABA. In electrophysiological studies, 3β5β-P a dual action on distinct positive and negative steroid modula- antagonized the 3α5β-P induced enhancement of GABA cur- tion sites associated with the GABAA-receptor (Park-Chung et al., rent (Maione et al., 1992; Maitra and Reynolds, 1998). On the 1999). other hand, 3β5α-P diminished the inhibitory effects of 3α5α- Consistent with the idea that PS induces activation-dependent P on population spikes evoked in rat hippocampal CA1 stratum inhibition on the GABAA-receptor (Wang et al., 2002), another pyramidal (Wang et al., 2000). Studies on the chloride uptake report revealed that the inhibition of basal inhibitory postsynap- into synaptosomes in rat cerebral cortex, in hippocampus and tic currents (IPSCs) by antagonist steroids was correlated with sIPSC in MPN showed that 3β-OH steroids reduced the 3α5α-P the basal decay time of the IPSCs (Eisenman et al., 2003). Analy- enhanced GABA response (Stromberg et al., 2006). In the pres- sis of single-channel behavior in the presence of GABA and PS ence of low concentration GABA, some of the 3β-OH steroids suggested no difference in the ability of PS to block liganded potentiated GABA-evoked chloride ion uptake and prolonged closed vs. liganded open receptors (Akk et al., 2001). These results the decay time, whereas the others had little or no effect on

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GABA stimulated current. Therefore, certain 3β-OH steroids, Selective antagonists of ρ receptors can prevent the development namely 5β-pregnane-3β, 20(S)-diol and 3β-OH-5β-pregnane-20- of myopia and enhance learning and memory in rats in the Mor- one have both agonistic and antagonist property (Wanget al.,2002; ris water maze task after intraperitoneal injection (Chebib et al., Stromberg et al., 2006). Moreover, another study with the Cl− 2009). Receptors containing the ρ subunit have been implicated in uptake method has shown that 3β-OH-5α-pregnan-20-one is a apoptosis of hippocampal neurons (Yang et al., 2003) and regula- useful antagonist of 3α5α-P enhanced GABA response (Lundgren tion of hormone release in the pituitary gland (Boue-Grabot et al., et al., 2003). However, in GABAA-receptor expressed in Xeno- 2000). pus oocytes showed that 3β-steroids inhibit GABA response at Several neurosteroids are shown to modulate the ρ1 recep- near-saturating concentrations (Rahman et al., 2006). It is still tor channel. Allopregnanolone, 5α-THDOC, and 5α-pregnane- unclear why the effect of 3β-OH steroids varies with different 3α-ol-11,20-dione (alphaxalone) potentiated the GABA-evoked methods. It is likely that the steroid structure, drug application currents from ρ1 receptor channels and concomitantly time and base line GABA concentration may be responsible for altered the deactivation kinetics by prolonging the decay the discrepancy. time. In contrast, pregnanolone, 5β-pregnane-3,20-dione (5β- It is suggested that a homologous mutation of the residue at dihydroprogesterone), and 5β-THDOC inhibited the GABA-  2 position closest to the cytoplasmic end of the M2 helix to serine elicited currents of the ρ1 receptor channel. In comparison to on both the β1 and the β2 subunit, α1V256S and β2A252S, reduced GABAA-receptors, the modulation of ρ1 receptor channels by the desensitization rate of GABA-activation at saturating doses neurosteroids occurred with relatively high concentrations and (Wang et al., 2007). In a receptor complex with reduced desensi- was more prominent in the presence of low concentrations of tization components to GABA-activation (e.g., mutant receptors), GABA. Structural comparison of these six neuroactive steroids the PS-inhibition was also greatly reduced (Akk et al., 2001). reveals that the key parameter in determining the mode of On the other hand, PS has been shown earlier to increase the modulation for the ρ1 receptor channel is the position of the deactivation rate of the GABA-evoked sIPSCs recorded by patch- hydrogen atom bound to the fifth carbon, imposing a trans- clamp techniques at the acutely dissociated neurons from the or cis-configuration in the backbone structure (Morris et al., MPN area of the rat brain slice (Haage et al., 2005). Recent 1999). studies confirmed the findings that PS increased the fast offset A study focusing on the electrophysiological effects of rate of GABA-activation (Wang et al., 2007). However, the slow inhibitory steroids on the ρ1 receptor found that steroid inhibitors component of the offset time course was decreased by PS in a could be divided into three major groups based on how mutations dose-dependent manner. The potencies of 5α-pregnan-3β,20α- to residues in the M2 transmembrane domain modified inhibition diol and 5β-pregnan-3β,20β-diol to influence offset time courses (Li et al., 2007). A recent study from the same research group (Li of GABA-activation were significantly lower than PS, in accor- et al., 2010) selected representatives of the three groups (preg- dance with our earlier findings while PS was significantly more nanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, potent to inhibit both peak and steady-state GABA currents than allopregnanolone sulfate and β-estradiol) to probe how these 3β-OH steroids (Wang et al., 2002, 2006, 2007). PS-inhibition was steroids, as well as the non-steroidal inhibitor picrotoxinin, mod- already seen at low dose of GABA response (≤EC20; Eisenman ify GABA-elicited fluorescence changes from the Alexa 546 C5 et al., 2003), whereas the inhibition by 3β-OH steroids on the cur- maleimide fluorophore attached to residues in the extracellular rent response was first seen at higher end (>EC50) of the GABA region of the GABAA-receptor. The fluorophore responds with dose-response curve (Wang et al., 2002). Obviously, 5α-pregnan- changes in quantum yield to changes in the environment, allow- 3β,20α-diol and 5β-pregnan-3β,20β-diol prolonged the fast offset ing it to probe for structural changes taking place during channel time course of GABA response,suggesting that the inherent associ- activation or modulation. The authors reported that the modula- ation between 3β-OH steroids and receptor has rather high affinity. tors have specific effects on fluorescence changes suggesting that However, it was not fully excluded that a more complicated model distinct conformational changes accompany inhibition (Li et al., of a multivalent interaction between hydrophobic steroids and 2010). Results are consistent with the steroids acting as allosteric receptor is employed. Kinetic properties of 3β-OH steroids have inhibitors of the ρ1 GABA receptor and support the hypothe- also been elucidated in acute dissociate neurons from the MPN sis that divergent mechanisms underlie the action of inhibitory area of the hypothalamus (Stromberg et al., 2006). These steroids steroids on the ρ1 GABA receptor. have no effect on the activation phase or the maximum amplitude of 3α5α-P enhanced IPSCs. They rather affect the slow deactiva- THE SITE OF NEUROSTEROID AGONIST ON THE tion phase. A recent report shows that 3β-OH steroids exert their GABAA-RECEPTOR effect by reducing the 3α5α-P induced prolongation of decay time Neurosteroids bind to GABAA-receptors at a site that is distinct constant (tdecay; Stromberg et al., 2006). from the recognition sites for GABA, benzodiazepines, and bar- biturates. This results in allosteric modulation of GABA bind- DIFFERENTIAL MODULATION OF ρ RECEPTOR CHANNELS BY ing or channel gating. The ability of neurosteroids to potentiate NEUROSTEROIDS GABA-activated currents recorded from a variety of neurons indi- The ρ1 GABA receptor is constitutes a dominant inhibitory force cated that most isoforms of the GABAA-receptor should be capa- in the retina but is expressed at lower levels throughout the ner- ble of binding neurosteroid modulators (Mitchell et al., 2008). vous system. Drugs acting on the ρ1 receptor can be useful in This concept was supported by studies of recombinant GABAA- the treatment of a variety of visual, sleep, and cognitive disorders. receptors with differing subunit composition that revealed a wide

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spectrum of activity for neurosteroids at GABAA-receptors with α-subunit. The importance of this site for neurosteroid binding some minor variations in efficacy (Puia et al., 1993; Lambert was also confirmed by the functional data on mutant α1 subunit et al., 1996; Maitra and Reynolds, 1999; Belelli et al., 2002). Neu- that reduced the potentiation of GABA responses by many syn- rosteroid modulation lacking subunit selectivity suggested that thetic steroids at α1β2γ2L receptors (Akk et al., 2008; Li et al., neurosteroids are binding to a site that is conserved throughout 2009). most members of the GABAA-receptor family. Studies using GABAA/glycine-receptor chimeras suggested an THE SITE OF NEUROSTEROID ANTAGONIST ON THE allosteric action of neuroactive steroids at the N-terminal side GABAA-RECEPTOR of the middle of the second transmembrane domain (M2) of It is generally accepted that the sulfate moiety is critical in pro- the GABAA-receptor β1 and/or α2 subunits (Rick et al., 1998). ducing a steroid that blocks rather than potentiates GABAA- Electrophysiological studies have confirmed that neurosteroid receptors (Park-Chung et al., 1999). The fact that an anionic agonists enhance Cl−-currents by increasing both channel fre- group is critical gave suggestion to us that the sulfate might actu- quency and channel open duration at GABAA-receptor (Callachan ally interact with residues forming the binding site mediating et al., 1987; Puia et al., 1990; Zhu and Vicini, 1997). Neurosteroid antagonism. However, no voltage dependency was observed with agonists do not require direct aqueous access to the receptor, pregnenolone sulfate-inhibition on the GABA response (Majew- and membrane accumulation is required for receptor modula- ska and Schwartz, 1987; Majewska et al., 1988; Akk et al., 2001), tion (Akk et al., 2005). Hosie et al. (2006) identified two dis- which suggests that the charged sulfate moiety does not interact crete binding sites in the receptor’s transmembrane domains that significantly with the membrane field as PS approaches the transi- mediate the potentiating and direct activation effects of neuros- tion state between unbound and unblocked to bound and blocked teroid agonists at the GABAA-receptor. The potentiating effect (Woodhull, 1973). Therefore, the lack of voltage dependence of of 3α5α-THDOC is mediated by a cavity formed by the α- pregnenolone sulfate-inhibition suggests that it is unlikely that subunit transmembrane domains. On the other hand, the direct the sulfate moiety interacts with a site deep within the channel activation of GABAA-receptorby3α5α-THDOC is mediated by (Akk et al., 2001). Results from our earlier report suggest that interfacial residues between α and β-subunits, and is enhanced pregnenolone sulfate was a γ2-subunit independent inhibitor at by steroid binding to the potentiation site (Hosie et al., 2006). GABAA-receptors (Wang et al., 2006). However, residues deep  These profiles indicate that two distinct neuroactive steroid bind- in the channel at the 2 position in the M2 helix of both α1- ing sites may exist; αTHr236 and βTyr284 residues in the trans- and γ2-subunit were critical for pregnenolone sulfate-inhibition membrane domain initiate direct activation whereas αGln241 (Akk et al., 2001; Wang et al., 2002, 2006). Theoretically, a large, and αAsn407 mediate the potentiating response (Hosie et al., rigid molecule such as a steroid would bind end-on rather than 2006). The neurosteroid potentiation site was further identi- with its long axis across the channel. It is thus possible that fied in the α1β2γ2S receptor by mutation of Q241 to methio- either the A-ring (where the sulfate moiety is attached) or the nine or leucine, which reduced the potentiation of GABA cur- D-ring (the other end of the molecule) would penetrate the Cl− rents by the naturally occurring neurosteroids, allopregnanolone, channel most deeply. The lack of voltage dependence indicates or tetrahydrodeoxycorticosterone (THDOC; Hosie et al., 2006, that the A-ring is unlikely to penetrate to the 2 position. The 2007). structure of the D-ring of PS is identical to that for many 3β- In order to address whether the potentiation site for neu- hydroxysteroids. Since the inhibitory properties of 5α-pregnan- rosteroids on GABAA-receptors is conserved amongst different 3β,20α-diol and 5β-pregnan-3β,20β-diol were also reduced GABAA-receptor isoforms, Hosie et al. (2006) used chimera to in the α1Mβ2γ2L receptor, we think it is more likely that the generate 100 models based on an alignment of the transmem- uncharged portions of the steroid molecules interact with the 2 brane domains of the GABAA-receptor α1 subunit and the nACh residue closest to the cytoplasmic end of the M2 helix on the α1 receptor α chain and selecting the 10 best models, revealed that subunit. the potentiation site is associated only with the a subunit and the The site of action of sulfated neuroactive steroids on GABAA- activation site is interfacial between the α- and β-subunit. receptors remains unclear. Based upon the observation that PS 35 By using heterologous expression of GABAA-receptors in HEK reduced the apparent affinity of [ S]-TBPS, Sousa and Ticku cells, in combination with whole-cell patch-clamp recording (1997) suggested that PS and DHEAS might bind at the picro- methods, a relatively consistent potentiation by allopregnanolone toxin/cage convulsant site. However, a mutation to the transmem- of GABA-activated currents was evident for receptors composed brane M2 channel domain eliminated picrotoxin sensitivity but of one a subunit isoform (α2–5) assembled with β3 and γ2S sub- the inhibitory effects of PS and DHEAS persisted (Shen et al., units (Hosie et al.,2009). By introducing mutant αβγ receptors,the 1999; Gibbs et al., 2006). The absence of voltage sensitivity or neurosteroid potentiation was dependent on the conserved gluta- alteration of single-channel open time argues against a binding mine residue in M1 of the respective a subunit (Hosie et al., 2009). site within the pore. Akk et al. (2001) identified a valine residue Studying wild-type and mutant receptors composed of α4β3δ sub- in the channel domain of the α1 subunit that slowed the devel- units revealed that the d subunit is unlikely to contribute to the opment of PS-inhibition when mutated to serine, but concluded neurosteroid potentiation binding site and probably affects the that this residue is unlikely to be part of the binding site and efficacy of potentiation (Hosie et al., 2009). likely influences PS action indirectly (Akk et al., 2001). Using In summary, the neurosteroid potentiation site was likely con- homologous mutation of the residue at 2 position closest to the tained entirely within or on the transmembrane domains of the cytoplasmic end of the M2 helix to serine on both α1 and β2

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subunit, namely α1V256S and β2A252S, it was found that effect the follicular phase and up to 4 nmol/L in the luteal phase. of PS is greatly reduced in these two mutant (Akk et al., 2001; In the third trimester of a pregnancy, serum concentrations of Wang et al., 2006, 2007). This suggests that these two amino allopregnanolone can increase to more than 100 nmol/L (Bicikova acids are involved in the PS mediated inhibition. However, it is et al., 1995; Luisi et al., 2000). Pregnanolone displays a similar pat- unclear whether these specific mutations at the cytoplasmic end tern with increase in the luteal phase (Wang et al.,1996; Sundstrom of M2 helix exert their effect by altering the allosteric mecha- et al., 1998). nism or by directly altering a binding site. A recent study in C. GABA-steroids are positive modulators of GABA responses elegans identified multiple residues in transmembrane domain 1 on the GABAA-receptor (Majewska et al., 1986). Chemically (M1), as well as a residue near the extracellular end of the M2 they are 3α-hydroxy-5α/β metabolites of progesterone (preg- helix, that are critical for low-micromolar inhibition of C. ele- nanolone and allopregnanolone), testosterone (3α 5α-androstan- gans GABAA-receptors by PS (Wardell et al., 2006). This latter diol),and desoxycorticosterone (tetrahydro-desoxycorticosterone, residue is of particular interest, as it is a positively charged argi- THDOC). GABA-steroids enhance the GABA effect on chloride nine that could potentially coordinate with the negatively charged flux resulting in increased inhibition of neural activity (Majewska sulfate of PS. The C. elegans receptor exhibits some pharmaco- et al., 1986; Gee et al., 1987; Birzniece et al., 2006b). GABA- logical differences as compared to mammalian GABAA-receptors steroids induce sedation and can be used as anesthetic drugs (for example, pregnanolone is inhibitory). So, these results may in human (Carl et al., 1990; Timby et al., 2006). GABA-steroids or may not be relevant to mammalian receptors; however, it is are anti-epileptic (Backstrom et al., 1984; Landgren et al., 1987) notable that an arginine residue is also found in this region of and in animal experiments they also possess an anxiolytic effect mammalian GABAA-receptor subunits (Gibbs et al., 2006; Wardell similar to benzodiazepines (Wieland et al., 1991). An anxiolytic et al., 2006). effect of allopregnanolone has, however, never been shown in Block of responses to high-efficacy agonists by this sulfated humans. steroid is greater than block of responses to partial agonists at sat- Human and animal studies revealed typical GABAA-receptor urating concentrations. This is called “activation dependant” or agonistic effects of allopregnanolone and pregnanolone at high “state dependant inhibition” (Wang et al., 2002; Eisenman et al., doses, i.e., sedation/anesthesia (Carl et al., 1990; Timby et al., 2003). Picrotoxin, another GABA channel blocker superficially 2006), anti-epileptic effects (Landgren et al., 1998), and anxiolytic similar to pregnenolone sulfate in its activation dependence but effects in animals (Wieland et al., 1991). However, earlier reports the site of action of PS does not require a functional picrotoxin from human and animal models also indicated that all GABAA- site for inhibition of GABA responses (Shen et al., 1999). The receptor agonists could induce negative symptoms with anxiety, GABA receptor antagonist Zn2+ also acts in activation dependant irritability/aggressiveness in certain individuals. Strong irritabil- manner. However, the Zn2+ binding site is located in the interface ity/aggression was induced in 3–6% of individuals and moderate between the α- and β-subunit and Zn2+ activity is minimal in symptoms are induced in 20–30% (Masia et al., 2000; Weinbroum ternary αβγ receptor (Hosie et al., 2003). et al., 2001). Interestingly, the prevalence of PMDD among women in reproductive age was in the similar range of 3–8%. The corre- GABA-STEROIDS AND THE MOOD CHANGE sponding prevalence of PMS, with milder symptom severity than Mood disorders are common health problems affecting women, PMDD, was 25–35% of women in reproductive age (Sveindottir especially during fertile ages. It is suggested that periods of hor- and Backstrom, 2000). monal variability, i.e., menarche (Angold et al., 1999), premen- It is puzzling why an increase in allopregnanolone during strual periods (Soares et al., 2001), postpartum (Kendell et al., the menstrual cycle is related to development of negative mood 1981; Chaudron et al., 2001), and perimenopause (Freeman et al., as allopregnanolone should be anxiolytic agent like benzodi- 2004) increase the risk of mood disorders in certain women. azepines. The answer depends on the fact that all GABAA- There are three obvious examples in interaction between mood, receptor agonists such as benzodiazepines, barbiturates, alco- steroids, and CNS, namely PMDD, side effects of oral contracep- hol, and allopregnanolone have paradoxical anxiogenic effects tives and negative mood symptoms encountered during sequential in certain individuals. At low concentrations or doses they progestagen addition to estrogen treatment in postmenopausal give severe adverse emotional reactions in a subset of indi- women. viduals (3–6%) and moderate reactions in up to 20–30% of An obvious relation between sex steroids and mood changes individuals. This paradoxical effect is induced by allopreg- are cyclic symptoms related to the menstrual cycle. Estradiol nanolone (Beauchamp et al., 2000; Miczek et al., 2003)ben- and progesterone display regular predictable changes during the zodiazepines (Ben-Porath and Taylor, 2002), barbiturates (Lee menstrual cycle. In parallel with the increase of progesterone, an et al., 1988; Kurthen et al., 1991; Weinbroum et al., 2001), and increase also occur in serum concentrations of allopregnanolone ethanol (Cherek et al., 1992; Dougherty et al., 1996; Miczek and pregnanolone (Wang et al., 1996). Although allopregnanolone et al., 2003). Symptoms induced by these GABAA-receptor active is synthesized in the brain, the major source to its brain con- drugs are depressive mood, irritability, aggression, and other centration is the corpus luteum of the ovary (Bixo et al., 1997; symptoms known to occur during the luteal phase in women Ottander et al., 2005). Allopregnanolone and progesterone are with PMS/PMDD. A biphasic effect was also observed for produced in parallel at the luteal phase of menstrual cycle (Wang medroxyprogesterone (MPA) and natural progesterone in post- et al., 1996; Genazzani et al., 1998). In fertile women plasma menopausal women taking hormone therapy. These women felt levels of allopregnanolone are approximately 0.2–0.5 nmol/L in worse on a lower dosage of MPA or progesterone than on a

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higher dosage or placebo (Bjorn et al., 2002; Andreen et al., 2005, reported that the change in effect coincide with an higher expres- 2006). sion of the α4β2δ subunit combination (Shen et al., 2007). The Thus allopregnanolone seems to have a biphasic effect on mood GABAA-receptor subunit combination that contains the δ-subunit with an inverted U-shaped relationship between concentration was known to be very sensitive to GABA-steroids, and is activated and effect. In postmenopausal women receiving progesterone, a by lower concentrations of allopregnanolone than other receptor biphasic relation between the negative mood symptoms and the subtypes (Wohlfarth et al., 2002; Liang et al., 2004). Therefore, plasma concentrations of allopregnanolone was observed. The the action of allopregnanolone at the α4β2δ receptor provides a negative mood increased with the elevating serum concentration mechanism for the generation of negative mood at puberty. The of allopregnanolone up to the maximum concentration seen at the proposed hypothesis requires a higher number or proportion of luteal phase. With further increase in allopregnanolone concentra- GABAA-receptors with the type(s) that are inhibited by steroids tion there was a decrease in symptom severity (Andreen et al., also in other conditions when paradoxical steroid effects are seen 2005, 2006). An inverted U-shaped relation between allopreg- (PMDD/PMS). Actually, the α4βδ subunit composition was a key nanolone dosage and irritability/aggression has also been noted factor in the progesterone withdrawal model for PMDD (Gallo in rats (Miczek et al., 2003). and Smith, 1993; Smith et al., 1998). A third hypothesis is that the paradoxical effect arises when POSSIBLE EXPLANATIONS FOR THE PARADOXICAL EFFECT the Cl− gradient across the membrane of critical neurons favors OF GABA-STEROIDS an excitatory rather than inhibitory effect of GABAA-receptor In this section, possible mechanisms of the biphasic response activation. The Cl− gradient across the cell membrane is subject to curve of allopregnanolone on behavioral parameters are discussed. active regulation and may also vary between different types of neu- The basic idea of so called paradoxical effect where neuros- rons as well as between different compartments within the neuron. teroids show one type of effect at low concentrations and another In adult vertebrate neurons, the intracellular Cl− concentration type at high concentrations is that an enhanced GABAA-receptor was usually relatively low and, the GABAA-receptor activation activity may give an excitatory net effect in certain situations, inhibited neuronal impulse activity. In contrast, during fetal and instead of the usual inhibitory effect (Backstrom et al., 2011). early postnatal development, the intracellular Cl− concentration The following hypotheses suggest several possible mechanisms was comparably high, and thus the GABAA-receptor activation how this can be achieved. In addition, there are no contradic- caused an excitation (Kahle et al., 2008). The intracellular Cl− tions between the different hypothesis and they may very well act concentration is determined not only by passive flux through the − in parallel. GABAA-receptor and other Cl -permeable channels, but also by One explanation is based on that inhibitory neurons are the cation-Cl−-cotransporters, with the major inward transport often most sensitive to GABA-steroids and therefore are the first to be mediated by NKCC1 and the major outward transport by KCC2 inhibited at elevated steroid concentrations. This will give a release (Price et al., 2005, 2009; De Koninck, 2007). In adults, the out- of the inhibitory tone at excitatory neurons, a so called disin- ward transport mediated by KCC2 dominates, which keeps the hibition. The idea emphasizes that specific combination of sub- intracellular Cl− concentration low. units determines the receptor sensitivity to neurosteroids (Belelli However, the intracellular Cl− concentration may be higher et al., 2002; Stromberg et al., 2006). In this context, extrasy- also in the adult brain, which has been demonstrated for the naptic GABAA-receptors containing the δ-subunit are of interest axon initial segment of cortical neurons (Szabadics et al., 2006; because they seem to be more sensitive to GABA-steroids than Khirug et al., 2008) and certain types of neurons in amygdala other types of subunit combinations (Mody, 2008). A possibil- (Martina et al., 2001) and substantia nigra (Gulacsi et al., 2003), as ity exists that one type of inhibitory neurons contains GABAA- well as in gonadotropin-releasing hormone neurons (Moenter and receptors with the δ-subunit, while the next in order contains DeFazio, 2005) and in presynaptic terminals (Haage et al., 2002). another subunit, making them less sensitive to GABA-steroids. Interestingly, estradiol was one factor that increased the activ- It has also been reported that the receptor subunit composition ity of NKCC1 under normal physiological conditions and thus and sensitivity to GABA-modulators can be regulated by environ- increased the intracellular Cl− concentration (Nakamura et al., mental factors such as stress (Biggio et al., 1990; Concas et al., 2004; Galanopoulou, 2008). Intriguingly, estradiol dose depen- 1996). dently increased the mood provoking effect of progestagens in Another idea is based on the finding that GABA-evoked cur- women (Bjorn et al., 2003), and negative mood effects in women rents at receptors with the α4β2δ combination are actually inhib- with PMDD/PMS (Dhar and Murphy, 1990). ited, instead of potentiated, by allopregnanolone (Shen et al., Finally, would it be possible to explain the inverted bell shaped 2007). During stress allopregnanolone and other GABA-steroids relationship between GABAA-receptor active steroid concentra- are produced and give an anxiolytic effect (Bitran et al., 1999). tions and symptoms that has been reported from clinical studies? However, Smith and colleagues showed that female mice reacted ThereportofPrescott et al. (2006) suggested that at a slightly with increased anxiety to stress during puberty (Shen et al., 2007). more positive Cl− equilibrium potential, a moderate increase in In humans, puberty is often a period characterized by mood GABAergic activity gave an increased excitation, whereas a larger swings and anxiety. This research group revealed that allopreg- GABAergic activity gave the opposite effect. However, whether an − nanolone changed from being a positive modulator of the GABAA- altered intracellular Cl concentration in adulthood may explain receptor, at the time before and after puberty, to become a negative the paradoxical effect of GABAA-receptor modulators remains to modulator at the time of puberty (Shen et al., 2007). They also be proven.

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THE ROLE OF NEUROSTEROIDS IN BEHAVIORAL DISORDERS magnocellular nucleus enhanced memory performance, whereas A number of review articles have discussed the important role allopregnanolone disrupted memory (Mayo et al., 1993). Preg- of neurosteroids in treating behavioral disorders by interacting nenolone, DHEA, and DHEAS increased memory when injected with the GABAA-receptor (Majewska, 1992; Reddy and Kulkarni, systemically, centrally or into the amygdala (Flood et al., 1988, 2000; Zorumski et al., 2000; Rupprecht et al., 2001). There are 1992; Wolkowitz et al., 1995). There was evidence that the con- a few obstacles preventing the clinical use of endogenous neu- centrations of DHEA and DHEAS decreased in patients suffering rosteroids. First of all, naturally occurring neurosteroids such as from AD (Sunderland et al., 1989; Nasman et al., 1991; Hillen et al., allopregnanolone have low bioavailability because they are rapidly 2000). It is promising that certain neurosteroids should be further inactivated and eliminated by glucoronide or sulfate conjuga- explored in the context of prevention and treatment of Alzheimer’s tion at the 3α-hydroxy group. The second obstacle is that the disease and mild cognitive impairment. 3α-hydroxy group of allopregnanolone may undergo oxidation to a ketone, restoring its activity at nuclear hormone receptors GABA-STEROIDS AND COGNITIVE FUNCTIONS (Rupprecht et al., 1993). Ganaxalone (3α-hydroxy-3β-methyl-5α- Chronic stress, stress-steroids, and sex steroids are linked to the pregnane-20-one), the 3β-methyl analog of allopregnanolone, is development of dementia (Shumaker et al., 2003; Lupien et al., an example of synthetic neurosteroid that overcomes these limi- 2005; Sandstrom et al., 2005). In women taking postmenopausal tations (Carter et al., 1997). Like allopregnanolone, ganaxalone is hormone therapy by administration of medroxyprogesterone, a a positive allosteric modulator of GABAA-receptor (Carter et al., GABA-steroid precursor, the risk for dementia, and AD doubled 1997). after 5 years of treatment (Shumaker et al., 2003). Medroxyprog- Neurosteroids modulate anxiety and stress level. After an acute esterone can induce anesthesia by acting on the GABAA-receptor stress stimulus, release of progesterone, pregnenolone, allopreg- (Meyerson, 1967). On the other hand, estrogen has been claimed nanolone, and 3α5α-THDOC occur in the blood circulation to protect against dementia (Henderson et al., 1994). This was, (Purdy et al., 1992; Barbaccia et al., 1998; Serra et al., 2000). Allo- however, not confirmed in the large Women’s Health Initiative pregnanolone and 3α5α-THDOC have potent anxiolytic activity (WHI) study but estrogen by itself did not increase the risk to in several animal anxiety models (Crawley et al., 1986; Bitran develop dementia (Shumaker et al., 2004). Hormone therapy with et al., 1995; Wieland et al., 1995; Reddy and Kulkarni, 2000). medroxyprogesterone resembles the exposure to stress-steroids However, the anxiolytic effect of allopregnanolone has not been during chronic stress. GABAA-receptor agonists are known to shown in human (Wihlback et al., 2006). Recent reports indicated impair memory. Allopregnanolone can inhibit learning in rats that allopregnanolone can induce aggression and anxiety at low tested in Morris water maze, an accepted test model for learning concentrations (Fish et al., 2001; Gulinello et al., 2001; Miczek and memory (Johansson et al., 2002). It is well-known that most et al., 2003). Allopregnanolone and cortisol levels are increased GABAA-receptor agonists, e.g., benzodiazepines (Barker et al., during the examination of PhD students (Droogleever Fortuyn 2004), barbiturates (Mohammed et al., 1987), and alcohol (Saun- et al., 2004). Therefore, it is suggested that allopregnanolone has ders et al., 1991; Vincze et al., 2007) impair memory and learning biphasic effects in certain individuals (Miczek et al., 2003). At low and increase the risk for permanent damages, although the risk doses it has an adverse, anxiogenic effect. This effect decreases with with low and moderate alcohol consumption is under debate increasing doses of allopregnanolone and the beneficial and calm- (Solfrizzi et al., 2007). ing property occurs (Beauchamp et al., 2000; Fish et al., 2001). The brain and serum concentrations of GABA-steroids vary A major concern with potential new anxiolytics is whether they with the production of adrenals, ovaries, and testicles (Purdy suffer the same drawbacks as classical benzodiazepines. Using the et al., 1991; Wang et al., 1996; Bixo et al., 1997; Luisi et al., elevated plus-maze paradigm for assessing the anxiolytic activity, 2000). Interestingly, classical stress hormones, cortisol, and cor- selective effects of neurosteroids have been reported which differ ticosterone, metabolized in a similar way and allotetrahydrocor- from diazepam (Rodgers and Johnson, 1998). Synthetic deriv- tisol (Allo-THF, 3α-hydroxy-5α-cortisol) interacts also with the ative Co 2-6749 30, which retains a 3β-trifluoromethyl group GABAA-receptor (Celotti et al., 1992). Allo-THF enhances the that should block metabolism and enhance oral bioavailability, inhibitory effect of allopregnanolone and has synergic effect on is selected for clinical development because there is a large sep- neuronal inhibition together with allopregnanolone (Stromberg aration between anxiolytic effects and side effects (Gasior et al., et al., 2005). That is why neurons are exposed to a stronger 1999; Vanover et al., 2000). Women with PMDD show often dif- inhibition during acute and chronic stress. During acute stress, ficulties in concentration and develop fatigue during the luteal allopregnanolone and THDOC increase in the brain of normal phase of the menstrual cycle, which is associated with high cir- animals. In animals subjected to chronic long-term stress, allo- culating levels of allopregnanolone (Sundstro and Backstrom, pregnanolone concentrations decreased in cortex at rest. However, 1999). a larger increase of allopregnanolone was observed in cortex Enzymes involving the production of allopregnanolone are after acute stress in this group of animals than normal con- present in the hippocampus (Compagnone and Mellon, 2000). trols (Serra et al., 2000). Patients with AD show an increased Using the Morris water maze paradigm, allopregnanolone was glucocorticoid production compared to healthy elderly control found to inhibit learning (Johansson et al., 2002). Antagonist subjects. In addition, an increased 5α-reduction was seen in neurosteroid 3β,20β-dihydroxy-5α-pregnane (UC1011), reduced patients with AD. Thus an increased glucocorticoid production the negative effect of allopregnanolone on learning in the Morris can be regarded as an early feature of AD since an enhanced water maze (Turkmen et al., 2004). PS infused into the basal production of 5α-reduced metabolites of cortisol was established

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(Rasmuson et al., 2001). 3α-OH-5α-reduced metabolites of cor- noradrenaline release in response to interleukin-1β (IL-1β) in late tisol interact with the GABAA-receptor and enhance the effect pregnancy (Brunton et al., 2009). Allopregnanolone signals the of GABA-steroids on the GABAA-receptor (Stromberg et al., pregnancy status of the animal to the brain and stimulates opi- 2005). oid production in the brainstem. Allopregnanolone also act via Chronic stress can impair memory (de Quervain et al., 1998). GABAA-receptors to enhance GABA action in the PVN. In rat, Memory impairment is permanent in persons with a chroni- allopregnanolone concentration increase greatly in both the cir- cally elevated adrenal production of GABA-steroids (Lupien et al., culation and the brain in pregnancy and reach a peak on day 2005). Memory impairment was also reported in chronic stress 19–20 of pregnancy (Concas et al., 1998; Brunton et al., 2009). syndromes, so called “burn-out syndrome.”“Burn-out syndrome” Parvocellular corticotropin-releasing hormone (CRH) neurons in occurs frequent in patients with AD. The production of corti- the PVN are under direct inhibitory GABAergic control (Mik- sol and GABA-steroids increased in parallel during stress (Purdy los and Kovacs, 2002). 5α-reductase activity is increased in the et al., 1991; Serra et al., 2003; Droogleever Fortuyn et al., 2004). hypothalamus in late pregnancy (Brunton et al., 2009), suggest- During chronic stress, long-term exposure to GABAA-receptor ing that the capacity to produce allopregnanolone is increased in agonist results in similar changes after prolonged exposure to pregnancy. Allopregnanolone may act locally to enhance GABA benzodiazepine and alcohol. Long-term and enhanced activation action in the PVN or on inputs to the CRH neurons to attenuate of the GABAA-receptor is an important factor in of memory HPA axis responses to stress in late pregnancy. The importance impairment during stress. The response of cortisol and GABA- of allopregnanolone to inhibit HPA axis responses to stress in steroids to adrenal stimulation was similar in patients with AD later pregnancy has been tested by blocking its production using a as chronically stressed animals (Nasman et al., 1991, 1996). 5αR inhibitor, finasteride (FIN). Blocking allopregnanolone gen- Patients with mild Alzheimer’s disease have a high and non- eration with finasteride (FIN) at a dose shown to reduce brain suppressible production of cortisol and GABA-steroids (Nasman allopregnanolone content by up to 90% (Concas et al., 1998), et al., 1995). substantially restores HPA axis responses to systemically adminis- After long-term exposure to GABA-steroids, down-regulation tered IL-1β in late pregnant rats (Brunton et al., 2009). AP appears of the GABAA-receptorisexpected(Yu and Ticku, 1995b; Barnes, to depend upon the actions of endogenous opioids to exert its 1996). A malfunctioned GABAA-receptor can be an important suppressive effects on HPA axis activity (Brunton et al., 2009). factor in the pathogenesis of stress-induced depression, “burn- Allopregnanolone induces opioid tone (as revealed by naloxone out” syndrome and sex-steroid related depression (Drugan et al., treatment) over ACTH responses to immune challenge in vir- 1989; Wihlback et al., 2006). The down-regulation occurs at dif- gin rats (Brunton et al., 2009). Induction of inhibitory opioid ferent levels in a time dependent manner: (i) desensitization; (ii) tone over HPA axis responses to IL-1β by AP treatment occurs receptor internalization; (iii) receptor subunit degradation; (iv) within 20 h, consistent with the rapid increase in pENK-A mRNA altered expression of receptor mRNA (Barnes, 1996). Exposure to expression in the NTS that has been seen after IL-1β treatment an agonist of the GABAA-receptor may cause changes in recep- (Engstrom et al., 2003). The mechanism by which AP upregulates tor mRNA and induce changes of the GABAA-receptor subunit opioid expression in the NTS is not clear. However, interac- composition (Smith et al., 1998). In women with PMDD, reduced tion with GABAA-receptors is a possibility (Blyth et al., 2000) benzodiazepine, ethanol, and GABA-steroid sensitivities occur in as has been described for CRH and vasopressin gene expres- the luteal phase, but not in the follicular phase of the men- sion in the PVN (Bali and Kovacs, 2003) and for hypothala- strual cycle (Sundstrom et al., 1997, 1998; Nyberg et al., 2004). mic oxytocin gene expression at the end of pregnancy (Blyth In rodents, repeated administrations of GABA-steroids caused et al., 2000). It is possible that allopregnanolone-induced opi- tolerance development (Birzniece et al., 2006a) in terms of GABA- oid tone represents a global mechanism through which HPA steroid-induced inhibition on learning (Turkmen et al., 2006). axis responses to other stressful stimuli are restrained in late It is well-known that prolonged exposure to allopregnanolone pregnancy. This pregnancy adaptation is expected to protect the altered the function of α4 subunit and α1 subunit (Smith et al., fetuses from adverse early life programming by maternal glu- 1998). In fact, the α4β2γ2 receptor is less sensitive to steroid cocorticoids, although this protection is obviously incomplete modulation than the α1β2γ2 receptor (Belelli et al., 2002). In (Brunton and Russell, 2010, 2011). Withdrawal of these mech- an earlier study on tolerance development in rats, acute tol- anisms or failure to maintain them appropriately might pre- erance against allopregnanolone-induced anesthesia developed dispose the fetuses to disease in later life (Welberg and Seckl, after 90 min of exposure and change in GABAA-receptor α4- 2001). subunit expression was observed in thalamus (Birzniece et al., 2006a). ACKNOWLEDGMENTS The author thank Torbjörn Bäckström and David Haage for ALLOPREGNANOLONE AND SUPPRESSED HPA AXIS STRESS advices and critical reading of an early version of the manu- RESPONSES IN LATE PREGNANCY OF RAT script. This work was supported by the Swedish Research Council- In late pregnancy, hypothalamo-pituitary–adrenal (HPA) axis medicine (project 73p-15450), Insamlingsstiftelsen för medi- responses to stressful stimuli are suppressed (Brunton and Rus- cinsk forskning vid Umeå Universitet, Svenska läkaresällskapets sell, 2003). Up-regulated opioid peptide produced by nucleus forskare program, ALF medel from västerbottens lans landsting, tractus solitarii (NTS) neurons acts presynaptically on noradren- Magn. Bergvalls Stiftelse and the EU regional fund’s objective 1 ergic terminals in the paraventricular nucleus (PVN) to inhibit program.

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Frontiers in Endocrinology | Neuroendocrine Science October 2011 | Volume 2 | Article 44 | 22 Wang Neurosteroids and GABAA-receptor function

Yee, B. K., Hauser, J., Dolgov, V. V., the GABA receptor subunits J. Pharmacol. Exp. Ther. 275, Received: 05 July 2011; paper pending Keist, R., Mohler, H., Rudolph, mRNA levels in mammalian cul- 784–789. published: 25 July 2011; accepted: 14 U., and Feldon, J. (2004). GABA tured cortical neurons following Zhu, W. J., and Vicini, S. (1997). Neu- September 2011; published online: 04 receptors containing the alpha5 chronic neurosteroid treatment. rosteroid prolongs GABAA channel October 2011. subunit mediate the trace effect Brain Res. Mol. Brain Res. 41, deactivation by altering kinetics of Citation: Wang M (2011) Neu- in aversive and appetitive condi- 163–168. desensitized states. J. Neurosci. 17, rosteroids and GABA-A receptor tioning and extinction of condi- Yu, R., and Ticku, M. K. (1995a). Effects 4022–4031. function. Front. Endocrin. 2:44. doi: tioned fear. Eur. J. Neurosci. 20, of chronic pentobarbital treatment Zorumski, C. F., Mennerick, S., Isen- 10.3389/fendo.2011.00044 1928–1936. on the GABAA receptor com- berg, K. E., and Covey, D. F. (2000). This article was submitted to Frontiers Yeung, J. Y., Canning, K. J., Zhu, G., plex in mammalian cortical neu- Potential clinical uses of neuroactive in Neuroendocrine Science, a specialty of Pennefather, P., MacDonald, J. F., rons. J. Pharmacol. Exp. Ther. 275, steroids. Curr. Opin. Investig. Drugs Frontiers in Endocrinology. and Orser, B. A. (2003). Tonically 1442–1446. 1, 360–369. Copyright © 2011 Wang. This is an open- activated GABAA receptors in hip- Yu, R., and Ticku, M. K. (1995b). access article subject to a non-exclusive pocampal neurons are high-affinity, Chronic neurosteroid treatment Conflict of Interest Statement: The license between the authors and Frontiers low-conductance sensors for extra- decreases the efficacy of benzodi- author declares that the research was Media SA, which permits use, distribu- cellular GABA. Mol. Pharmacol. 63, azepine ligands and neurosteroids conducted in the absence of any tion and reproduction in other forums, 2–8. at the gamma-aminobutyric commercial or financial relationships provided the original authors and source Yu, R., Follesa, P., and Ticku, M. acidA receptor complex in that could be construed as a potential are credited and other Frontiers condi- K. (1996). Down-regulation of mammalian cortical neurons. conflict of interest. tions are complied with.

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