The GABAR [GABAA (γ-aminobutyric acid type A) receptor], which mediates most inhibition in the brain, is regulated homoeostatically to maintain an optimal level of neuronal excitability. In particular, the α4βδ subtype of the GABAR plays a pivotal role in this regulation. This receptor, which is expressed extrasynaptically on the dendrites, normally has low expression in the brain, but displays a remarkable degree of plasticity. It can also be a sensitive target for endogenous neurosteroids such as THP (3α-hydroxy-5[α]β-pregnan-20-one (allo-pregnanolone); a neurosteroid and positive modulator of the GABAR), which is released during stress, although the effect of the steroid is polarity-dependent, such that it increases inward current, but decreases outward current, at α4β2δ GABAR. Expression of α4β2δ GABAR in CA1 hippocampus is also tightly regulated by fluctuating levels of neurosteroids, as seen at the onset of puberty. Declining levels of inhibition resulting from the decrease in THP at puberty are compensated for by an increase in α4βδ GABAR along the apical dendrites of CA1 hippocampal pyramidal cells, which reduces neuronal excitability by decreasing the input resistance. However, excessive decrease of neuronal function is averted when THP levels rise, as would occur during stress, because this steroid decreases the outward GABAergic tonic current via inhibition of α4β2δ GABAR, thereby restoring measures of neuronal excitability to pre-pubertal levels. Thus the homoeostatic regulation of α4βδ GABAR expression plays an important role in maintaining ambient levels of neuronal excitability at puberty.
The GABAR [GABAA (γ-aminobutyric acid type A) receptor]
The GABAR mediates most inhibition in the CNS (central nervous system). This receptor has a pentameric structure typically composed of two α, two β and one γ subunits , where the δ subunit can substitute for the γ . There are as many as six α, three β and three γ subunits, as well as ε, θ, π and ρ subunits, which can combine in different isoforms, yielding receptors with vastly different biophysical and pharmacological properties . Each subunit, in turn, comprises four TM (transmembrane)-spanning α-helices, where TM2 lines a central Cl− channel and the intracellular TM3–TM4 loop is the target for phosphorylation, as well as a regulator of receptor trafficking . GABA (γ-aminobutyric acid; an inhibitory neurotransmitter) binds to a selective site located in the cleft between the α and β subunits to gate a Cl− conductance, which, in most areas of the adult CNS, is inhibitory. In regions such as the hippocampus, GABA is hyperpolarizing and also produces a shunting inhibition . The direction (polarity) of the current is controlled by activity of the K–Cl− co-transporters KCC2 and NKCC1, which is outward (inward flux) in CA1 hippocampal pyramidal cells  and inward in dentate gyrus granule cells , both yielding inhibitory effects.
GABARs can be localized either subsynaptically, predominantly at the soma of the neuron, or extrasynaptically, where they can be localized perisynaptically or distant from inhibitory synapses [7,8]. Although multiple subtypes of GABAR can be found at either location, the γ subunit  is required for tethering at the synapse, where scaffolding proteins such as gephyrin play a role . In contrast, extrasynaptic receptors include α5β3γ2 , which shows high expression on CA1 hippocampal pyramidal cells, and α4β2δ , which shows high expression on dentate gyrus granule cells and thalamic relay neurons . Recent evidence also suggests that receptors containing the γ subunit can also localize extrasynaptically , as well as the α1β2 GABAR isoform . Of these receptor subtypes, the α4β2δ shows a relatively low expression across brain regions , but has a very high degree of receptor plasticity triggered by changes in the level of inhibitory tone [13–16].
GABA-gated current is responsive to a range of potent modulators, both endogenous and synthetic, which typically have sedative effects. These include the BDZ (benzodiazepine) class of compounds, which comprise agonists and inverse agonists that enhance or decrease GABA-gated current respectively, as well as selective antagonists . BDZ agonists bind in the cleft between the α and γ subunit; thus GABARs lacking a γ subunit are insensitive to BDZ modulation. In addition, the α4 and α6 subunits do not bind BDZ agonists, owing to an arginine-to-histidine modification at residue 100 . In addition to receptor subtypes that are completely insensitive to BDZ modulation, there are receptor subtypes that display atypical responses to BDZ ligands. α4βγ2 GABARs are not modulated by BDZ agonists, but are positively modulated by the BDZ antagonist flumazenil as well as the partial inverse agonist RO15-4513 . The differential response of these receptor subtypes to BDZ modulation can be used to identify altered receptor expression using electrophysiological or behavioural assays . Other modulators include barbiturates, ethanol, anaesthetics and neurosteroids, for which the binding site has recently been identified .
The δ-containing GABARs, α1β3δ and α4β2δ, are especially unique in their biophysical and pharmacological properties. These receptors have a high sensitivity to GABA [13,21] and little desensitization [21,22], making them ideally suited for their extrasynaptic location  where they come into contact with ambient GABA concentrations <1 μM from spillover of synaptically released GABA , which are regulated by GABA transporters . These receptors have a lower open channel probability and low efficacy state [25,26], but display a high sensitivity to modulators, including barbiturates , ethanol [13,27] and endogenous steroids [21,28] that increase receptor efficacy . Unlike the typical subsynaptic GABARs, δ-containing GABARs are primarily localized on the apical dendrites in regions such as dentate gyrus and CA1 hippocampus , where their impact would be expected to influence incoming afferent activity.
δ-Containing GABAR underlie a tonic current [29–31] in dentate gyrus, cerebellum and thalamus, which in many cases can be sensitive to modulation by neurosteroids such as THP (3α-hydroxy-5[α]β-pregnan-20-one (allo-pregnanolone); a neurosteroid and positive modulator of the GABAR) and THDOC (5α-pregnane-3α,21-diol-20-one; a neurosteroid and positive modulator of the GABAR), although conflicting reports have appeared. These steroids are metabolites of progesterone and corticosterone respectively and both are released after 30–45 min of stress . In addition, circulating levels of THP parallel those of progesterone, which fluctuate across the ovarian cycle, at puberty and during pregnancy.
α4 GABAR expression
What distinguishes the GABAR response to modulators is that homoeostasis of inhibitory control is maintained over long-term periods. That is, although the acute response can reveal potent modulation of the receptor, sustained receptor modulation leads to receptor plasticity that maintains inhibition at an optimal level. The level of inhibitory control is critical in allowing appropriate levels of response to afferent input in neuronal circuits without epileptic activity or decrease of neuronal output. Receptors containing the α4 subunit, namely α4βγ2 or α4β2δ, have recently been identified as chief mediators of this type of receptor plasticity [14,16,19]. In addition, under conditions when α6βδ-containing GABARs are not expressed in knockout animals, an adaptive increase in a voltage-independent K+ channel compensates for the lack of tonic inhibition in cerebellar granule cells , signifying the importance of maintaining this level of inhibition.
Sustained 48 h exposure to positive modulators of the GABAR, such as ethanol, BDZs, barbiturates and THP [33–36], increases the expression of the α4βγ2 GABAR, which substitutes for the normally highly expressed α1β2γ2 GABAR . The α4β2γ2 GABAR has a lower open probability and faster deactivation than α1β2γ2 [26,37–39], thus decreasing the level of inhibition to compensate for the increase in current produced by the GABA modulators, as suggested by studies showing that increased α4βγ2 expression decreases paired pulse inhibition in CA1 hippocampus . Recent in vitro findings  suggest, in fact, that the level of α4 expression is highly correlated with the change in GABA-gated current, where exposure to positive modulators increases α4 expression, while exposure to negative modulators decreases α4 expression and exposure to ligands, such as flumazenil, which bind, but produce no change in current, does not alter α4 expression. Functional expression of the α4β2γ2 GABAR was verified in these studies by its distinctive pharmacology , displaying insensitivity to BDZ agonists and where a BDZ antagonist and the partial inverse agonist, RO15-4513, act as BDZ agonists by increasing GABA-gated current. However, the initial trigger for this subunit ‘switch’ is dependent on the GABA-gated current, because reducing the Cl− gradient by inhibiting the KCC1 co-transporter prevented changes in α4 expression normally seen with positive GABA modulators .
Interestingly, withdrawal from sustained administration of positive GABA modulators, such as ethanol, BDZs and THP, via its parent compound progesterone, also increases α4 expression [19,41,42], yielding receptors pharmacologically consistent with expression of α4βγ2 GABAR . Time-course studies suggest that α4 expression is a transient outcome of both 48 h exposure and withdrawal states , whereas α4 expression returns to control levels after continuous exposure to THP. The reason for this complex response to continued inhibition is not clear, but may be the result of other compensatory mechanisms. A number of studies have suggested that modulators may switch their target neuron from principal cells to interneurons or from extrasynaptic to synaptic locations [43,44]. However, this remains to be determined in the case of neurosteroid regulation of GABAR expression.
Expression of the α4β2δ GABAR is also an outcome of fluctuating levels of inhibition produced by neurosteroids. Exposure to endogenous or exogenously administered steroids (THP or THDOC), across a timespan of 30 min to 48 h, increases the expression of α4βδ GABAR in areas such as CA1 hippocampus and dentate gyrus [35,45]. A number of reports suggest that this receptor is the most sensitive target for neurosteroids, although that may depend on other factors such as phosphorylation state .Thus the increase in the receptor target for these steroids would permit optimal effectiveness of these steroids.
However, after sustained exposure to THP, the withdrawal from this steroid-induced inhibition also increases the expression of α4βδ GABAR as a compensatory response to maintain optimal levels of inhibition . A number of studies have demonstrated increased expression of α4 and δ levels after THP ‘withdrawal’ in diverse areas, such as CA1 hippocampus [13,14], dentate gyrus [16,47] and periaqueductal grey . The increase in α4βδ expression was seen after exogenous administration of steroid as well as at parturition after elevated levels of THP produced during pregnancy or across steroid fluctuations produced by the ovarian cycle.
In the mouse, inhibitory tone in CNS circuits cycles in a circadian manner, due to robust nightly surges of THP . Thus ablation of this steroid by preventing its formation with finasteride, a 5α-reductase inhibitor, creates a withdrawal state from high, physiological CNS levels of the GABA-enhancing steroid . Under these conditions, α4 and δ subunits increase expression on CA1 hippocampal pyramidal cells . Functional co-expression of α4 and δ was determined using a pharmacological approach, as the GABA agonist gaboxadol [THIP (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol)] has increased potency at δ-containing GABAR when compared with other subtypes, while lanthanum uniquely inhibits current at α4βδ GABAR . Here, the increase in tonic inhibition produced by increased expression of these receptors would offset the loss of inhibition due to the loss of neurosteroid.
Puberty and α4βδ GABAR
The onset of puberty is a physiological state associated with marked increases in expression of α4βδ GABAR from almost undetectable levels before puberty . The expression of α4 and δ was detected along the apical dendrites of CA1 hippocampal pyramidal cells of female mice using immunocytochemical/electron microscopic techniques, quantified with Western-blot analysis and verified using pharmacological means, by assessing the responsiveness of the tonic inhibitory current to gaboxadol and lanthanum. Although many hormonal changes occur at the onset of puberty, the trigger for this increase in α4βδ GABAR is the decline in THP levels that occurs at this time . Administration of replacement THP for 48 h prevented the increase in this receptor population, as well as the ensuing increase in the tonic current it produced at puberty.
The increase in dendritic α4βδ receptor expression at puberty was evident as an increase in GABA conductance , which reduced the input resistance of the pyramidal cell, thereby increasing the current necessary for triggering an action potential (Figure 3). Thus the increase in α4βδ GABAR expression was a compensatory response to the reduction in inhibition produced by declining levels of THP at puberty.
Although the compensatory increase in inhibition at puberty maintains an optimal level of inhibition in the absence of THP, inhibitory tone would be increased excessively if THP levels were to rise. Because α4βδ GABAR is a sensitive target for THP , increased release of this steroid produced during stress would dampen the hippocampal circuitry beyond the optimal level and impair cognition. This outcome is avoided by the unique steroid responsiveness of the α4β2δ GABAR, which is polarity-dependent .
Polarity-dependent effect of THP
Studies using recombinant receptors transiently expressed in HEK (human embryonic kidney)-293 cells reveal that THP potentiation of current gated by α4β2δ GABAR is seen only under conditions where the current is inward (outward flux)  (Figure 1). However, under conditions where the current is outward, THP decreases the GABA-gated current. This polarity-dependent effect of the steroid was only observed in α4β2δ GABAR. Substitution of α1 for α4 or β3 for β2 yielded receptors that were potentiated by the steroid, regardless of current polarity (Figure 1). Other commonly expressed receptors, including α1β2γ2 and α5β3γ2, which have high expression on CA1 hippocampal pyramidal cells, did not display polarity-dependent steroid potentiation, nor did α4β2γ2 or α4β2. The steroid did not alter the reversal potential of the current, suggesting that it was not affecting other non-GABA conductances and was correlated with the concentration of GABA. A direct examination of receptor kinetics in response to rapid application of agonist revealed that the steroid accelerated desensitization as a potential mechanism for its reduction of outward current at α4β2δ  (Figure 1). Other studies have suggested that neurosteroids accelerate desensitization of δ-containing GABAR .
Effects of a neurosteroid at α4β2δ GABAR are polarity-dependent
In order to understand a mechanism for the polarity-dependent effect of THP at α4β2δ GABAR, it is important to consider the sequence homologies of α1 and α4, as α1β2δ did not display polarity-dependent effects of the steroid. In fact, the least homologous region of these sequences is within the TM3–TM4 intracellular loop : The α4 loop is 2-fold longer than the α1 loop, and has less than 10% sequence homology (Figure 2). Because positively charged amino acid residues have been identified as modulatory Cl− sites on other receptors, such as hCN (hyperpolarization-activated cation channel) , which mediates Ih (hyperpolarization activated current), we sequentially mutated these sites within the α4 TM3–TM4 loop. Mutation of Arg353 to a neutral leucine residue successfully prevented the polarity-dependent effect of the steroid , suggesting that it acts as a potential Cl−-binding site that is necessary and sufficient for steroid-induced decreases in outward current at α4β2δ GABAR (Figure 2).
Arg353 in the α4 subunit is necessary for the polarity-dependent inhibition of α4β2δ GABAR by THP
Modulatory effects of Cl− have been noted previously [51,52] that are necessary for barbiturate and BDZ enhancement of GABA binding. Recent studies suggest that ion-sensor sites can regulate other events such as Cl− activation of hCN subunits which mediate Ih . In addition, the recent discovery  of a cation-triggered phosphorylation event in a novel membrane protein lacking an ion pore suggests that ion-sensor sites regulate neuronal function beyond ion conductance. In addition, the intracellular loop of the cysteine-loop family of receptors is ion accessible [54,55], whereas for other membrane receptors this loop functions not only as a permeation pathway, but also as a site necessary for rapid desensitization .
GABAergic tonic current at puberty
Because the effect of the steroid was polarity-dependent, the direction of GABAergic current was investigated at the onset of puberty using tight-seal cell-attached recording in current clamp mode, which permits relatively accurate measurement of the membrane potential as the ratio of the seal resistance to the patch resistance becomes infinitely large . Using this technique, local dendritic application of the GABA agonist gaboxadol produced a downward deflection , signifying a hyperpolarizing potential on these cells at puberty. This effect was verified by recording the current using perforated patch techniques in voltage clamp mode where the internal Cl− milieu was undisturbed. Under these conditions, application of 30 nM THP produced a similar downward deflection as did a GABA antagonist, suggesting that the steroid reduced the GABAergic tonic current of CA1 hippocampal pyramidal cells at puberty. In contrast, in areas such as dentate gyrus, the GABAergic tonic current is inward , where THP would enhance the tonic inhibition .
The steroid-induced reduction in GABA conductance was evident as an increase in the input resistance , which reduced the current necessary for generating an action potential (Figure 3), thereby increasing neuronal excitability. THP did not change the reversal potential for the tonic current, nor did it have a significant effect on the tonic current recorded from hippocampi of pubertal mice lacking expression of the δ subunit. Taken together, these findings suggest that THP acts to reduce outward current at α4β2δ GABAR, which underlies a tonic current at puberty.
THP lowers the current threshold for spiking of pyramidal cells at the onset of puberty
These findings suggest that plasticity of GABAR changes at puberty are 2-fold: first, the increase in α4β2δ GABAR underlies a tonic inhibition, which compensates for the reduction in inhibition due to the reduction in ambient levels of the neurosteroid THP at the onset of puberty. Secondly, these receptors not only increase the inhibitory tone of the hippocampal circuitry, but are also designed to be inhibited by the stress steroid THP in areas of the brain, such as CA1 hippocampus, where the GABAergic current is outward. This would prevent an overly suppressive effect on the circuit when THP levels are increased by stressful events. However, in areas of the brain, such as the dentate gyrus, where the GABAergic tonic current is inward, THP effects consistently enhance inhibition. Thus this unique extrasynaptic receptor can normalize inhibition with individualized effects to regulate the degree of steroid inhibition appropriate for selective brain regions.
Neuronal Glutamate and GABAA Receptor Function in Health and Disease: Biochemical Society Focused Meeting held at University of St Andrews, St Andrews, U.K., 21–24 July 2009. Organized and Edited by Chris Connolly and Jenni Harvey (Dundee, U.K.).
This work was supported by the National Institutes of Health [grant numbers DA09618 and AA12958 (to S.S.S.)].