Associate editor: MorrowNeurosteroid regulation of GABAA receptors: Focus on the α4 and δ subunits
Introduction
Gamma-aminobutyric acid (GABAA) receptors (GABAR) mediate the majority of inhibition in the brain, essential for sculpting patterns of circuit activity, gating relevant sensory signals (Smith & Chapin, 1996), and restoring neuronal inhibition via feedback pathways (Smith, 2003). Behaviorally, these receptors influence mood, relieving anxiety states, and suppress seizure activity (Smith, 2003). The function of these receptors is determined to some extent by their subtype, which is under dynamic control (Mody, 2005) by the principal modulators of GABAergic inhibition in the brain, the neurosteroids.
The endogenous neurosteroid 3α-OH-5α-pregnan-20-one (allopregnanolone or 3α,5α-THP) and its active 5β isomer 3α,5β-THP (collectively referred to here as THP) are metabolites of the ovarian/adrenal steroid progesterone (Compagnone & Mellon, 2000). Similar compounds such as 5α-pregnane-3α,21-diol-20-one (THDOC) are metabolites of the adrenal steroid corticosterone (Compagnone & Mellon, 2000). Unlike classic steroids which act via nuclear receptors with ensuing genomic effects, THP and THDOC act selectively as potent positive modulators of the GABAR (Majewska et al., 1986, Callachan et al., 1987, Gee et al., 1987, Turner et al., 1989), increasing GABA-gated current at physiological concentrations by increasing the open duration and frequency of channel openings (Twyman & Macdonald, 1992). Potentiation of GABA inhibition has also been seen after intravenous administration of its parent compound progesterone (Smith et al., 1987).
The GABAR mediates most fast inhibition in the central nervous system (CNS; Hevers & Luddens, 1998). This receptor possesses remarkable structural diversity: composed of 5 subunits, the usual stoichiometry of the receptor is 2α, 2β and 1 γ or δ subunit (Chang et al., 1990), but multiple subtypes of subunits exist, creating a vast pool of possible receptor isoforms. These different subunit combinations result in receptors with strikingly different pharmacological and biophysical properties, sometimes resulting in distinctive functional effects on excitability of CNS circuits (Hevers & Luddens, 1998).
The localization pattern of GABAR subtypes reveals highest expression of α1β2γ2 throughout the CNS (Wisden et al., 1992), which can be localized at GABAergic synapses (Nusser et al., 1996) where saturating or near-saturating levels of GABA (1 mM) are released for brief exposures (< 1 ms). Alternatively, α1β2γ2 GABAR can be localized at extrasynaptic sites where they would come into contact with ambient levels of GABA (1 μM or less) (Wu et al., 2003). Other receptor subtypes have a heterogeneous distribution, with α5-containing GABAR localized exclusively to extrasynaptic sites on CA1 hippocampal pyramidal cells, where they underlie a tonic inhibition (Caraiscos et al., 2004). In contrast, α6-containing GABAR are found exclusively on cerebellar granule cells at both synaptic and extrasynaptic sites (Nusser et al., 1998).
The α4 subunit has relatively low expression in the CNS (Wisden et al., 1992), but is concentrated on dentate gyrus granule cells, thalamic relay neurons and to a lesser extent on neurons in cerebral cortex and striatum. This subunit can coexpress with either γ2 or δ (Sur et al., 1999), yielding receptors which are insensitive to modulation by benzodiazepines (BDZ; Wisden et al., 1991, Wafford et al., 1996, Benke et al., 1997) due to an arginine at residue 99. Mutation of the homologous arginine to histidine in the BDZ-insensitive α6, as found in BDZ-sensitive α subunits (i.e., α1, α2, α3, α5) reverses this effect (Wieland et al., 1992), yielding receptors which bind BDZs (Binkley & Ticku, 1991). Recent evidence suggests that α4βγ2 may be localized at the synapse (Hsu et al., 2003, Chandra et al., 2006), as well as extrasynaptically, as shown both by electron microscopy and pharmacological modulation of the tonic current (Liang et al., 2004, Liang et al., 2006).
In contrast, α4β2δ GABAR are exclusively extrasynaptic (Wei et al., 2003) because they lack the γ subunit. α4β2δ GABAR have a high sensitivity to GABA (1 μM = ∼ EC75; Sundstrom-Poromaa et al., 2002) but desensitize little (Brown et al., 2002, Feng et al., 2006), making them ideally suited for an extrasynaptic function. δ-containing GABAR are also the most sensitive to modulation by steroids (Wohlfarth et al., 2002, Brown et al., 2002, Belelli et al., 2002, Bianchi and Macdonald, 2003, Liang et al., 2004), which increase receptor efficacy (Bianchi & Macdonald, 2003). α4β2δ GABAR mediate a tonic inhibitory current in thalamus (Belelli et al., 2005, Cope et al., 2005, Jia et al., 2005) and dentate gyrus (Stell et al., 2003, Mtchedlishvili and Kapur, 2006), which is sensitive to modulation by steroids, such as THP (Mtchedlishvili & Kapur, 2006) and the related steroid THDOC (Stell et al., 2003, Cope et al., 2005). This tonic current is thus the most sensitive target for steroid-induced changes in inhibitory tone of neuronal circuits, as evidenced by the reduced steroid sensitivity of mice lacking expression of the δ subunit (Mihalek et al., 1999, Spigelman et al., 2003, Shen et al., 2007).
Several reports suggest that steroids such as THDOC increase current gated by δ-GABAR by increasing receptor efficacy (Bianchi et al., 2002), augmenting peak current but accelerating desensitization, to ultimately reduce current amplitude gated by saturating concentrations of GABA. However, recent studies have suggested that physiological concentrations of THP (30 nM) can accelerate the desensitization of α4β2δ GABAR in response to ambient concentrations of 1 μM GABA in a polarity-dependent manner (Shen et al., 2007). THP produced rapid desensitization of outward current (inward Cl− flux), but potentiated inward current at these receptors, suggesting that the ultimate effect of this steroid on neuronal excitability is in part determined by the direction of the Cl− gradient. In areas which normally have high levels of α4β2δ GABAR expression, dentate gyrus and cortex (Wisden et al., 1992), the GABAergic current is inward (Staley and Mody, 1992, Gulledge and Stuart, 2003), and thus inhibition in these areas would be enhanced by THP.
This polarity-dependent decrease in current generated by THP at α4β2δ GABAR was dependent upon a basic residue, arginine 353, in the intracellular loop of α4, which may function as a putative Cl− modulatory site (Shen et al., 2007). Polarity-dependent rates of desensitization have been reported for other GABAR, including the homologous α6 (Bianchi et al., 2002) as well as α5 (Burgard, 1996). This steroid-mediated polarity dependent decrease in inhibition would have important implications for effects of steroids across brain areas which generate outward GABAergic current via α4β2δ GABAR.
GABAR populations are not static, but rather undergo dynamic modulation when the balance of inhibition is increased in a chronic manner. This review will detail examples of plasticity in the GABAR population which result from fluctuations in naturally occurring or administered steroids such as THP. In particular, the α4 subunit undergoes marked changes in expression as a response to fluctuating levels of steroids. These steroid-induced fluctuations in GABAR isoforms result in alterations in CNS excitability with implications for syndromes such as pubertal mood swings, premenstrual syndrome (PMS), postpartum blues, and the perimenopause when alterations in mood can occur.
Section snippets
Steroid metabolism
THP is produced systemically from progesterone of ovarian or adrenal origin, but can also be synthesized de novo in the brain from cholesterol via side chain cleavage enzyme which forms pregnenolone, the precursor of all steroid molecules (Compagnone & Mellon, 2000). 3β-Hydroxysteroid dehydrogenase converts pregnenolone to progesterone, which is then converted via neuronal enzymes into THP. Both the 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSD) enzymes are under steroidal control (
Time course of steroid effects on α4 subunit expression
Because exposure to endogenous steroids across natural cycles is normally extended across days to weeks, a number of studies have investigated the effect of chronic steroid exposure on GABAR function. In our laboratory, in vivo treatment of female rats with progesterone or THP increased expression of the normally underexpressed α4 subunit in the hippocampus, an effect which reached significant levels after 48–72 hr (Fig. 1; Gulinello et al., 2001, Hsu et al., 2003). This was a transient event,
Effect of THP withdrawal on α4 subunit expression
As noted above, the timecourse of changes in α4 expression as a response to the continued exposure to THP is complex (Gulinello et al., 2001). In addition to its effect on GABAR plasticity after 48-hr exposure, THP also exhibits withdrawal properties following chronic exposure. Following a 21-day administration of either THP directly (Fig. 4) or its parent compound progesterone to female rats, discontinuation of steroid administration (i.e., “withdrawal”) resulted in a marked 3-fold increase in
GABAA receptor δ subunit
Several studies have correlated changes in GABAR subunit expression with the estrous cycle. In one landmark study (Maguire et al., 2005), increases in δ subunit expression on diestrus-1 were correlated with increased amplitude of the tonic current recorded from the dentate gyrus granule cells. Diestrus-1 is associated with a secondary peak of THP, and the δ-containing GABAR are the most sensitive targets for this steroid (Wohlfarth et al., 2002). Indeed, increased expression of δ was associated
Puberty
It is well-known that puberty is associated with mood swings and aversive responses to stress (Buchanan et al., 1992, Hayward and Sanborn, 2002, Modesti et al., 1994). Because puberty is also associated with fluctuations in circulating levels of THP (Fadalti et al., 1999), we examined the expression levels of α4 and δ subunits in CA1 hippocampus immediately before and after the onset of puberty. In fact the onset of puberty resulted in markedly increased expression of α4 and δ subunits along
Benzodiazepines
The neurosteroid-induced increase in α4 subunit expression may be linked to sustained increases in GABA-mediated inhibition, because α4 expression is regulated by other positive modulators of the GABAR. Indeed, chronic 7- or 14-day treatment with the BDZ diazepam or the α1-selective zolpidem increases α4 mRNA expression in rat cortex (Holt et al., 1996), in association with decreases in α1 expression. Other studies have indicated that withdrawal from chronic treatment with either diazepam or
Mechanisms of α4 expression
The mechanism for the 48-hr steroid-induced upregulation of α4 subunit expression has not been established, but it requires low background levels of steroid, and is most effectively observed after neuronal differentiation by nerve growth factor in a neuroblastoma cell line (Zhou & Smith, 2007). THP is not the only steroid which is associated with higher levels of α4-containing GABAR. Another steroid hormone, 17β-estradiol, increases α4 levels when administered across a 48-hr or 7-day exposure (
Puberty
Puberty is well-known as a time when mood swings occur (Hayward & Sanborn, 2002), and responses to stressful events become exacerbated (Modesti et al., 2004). Although an array of hormonally mediated events occur at this time, the onset of puberty is associated with a decline in circulating levels of THP (Palumbo et al., 1995, Fadalti et al., 1999). The onset of puberty also increases the likelihood of developing anxiety disorders, including panic disorder, twice as likely in girls compared to
Conclusions
Taken together, the results from these varied basic science and clinical studies suggest that beyond the acute effects of neurosteroids, chronic exposure to steroids such as THP and THDOC produce compensatory changes in target receptor populations. Notable among the subunits which are altered include the α4 and δ subunits. Increases in α4 expression would produce BDZ insensitivity, one common factor in rodent models and clinical examples of PMDD. Depending on the direction of Cl− current in
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