GHB receptor targets in the CNS: Focus on high-affinity binding sites

Biochemical Pharmacology 87 (2014) 220–228

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Commentary

GHB receptor targets in the CNS: Focus on high-affinity binding sites Tina Bay 1, Laura F. Eghorn 1, Anders B. Klein, Petrine Wellendorph * Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 August 2013 Accepted 29 October 2013 Available online 20 November 2013

g-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and

Keywords: GHB receptor GABAA receptor GABAB knock-out mice NCS-382

high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain. ß 2013 Elsevier Inc. All rights reserved.

1. Introduction to GHB: Neurogenic substance, therapeutic drug and drug of abuse

g-Hydroxybutyric acid (GHB) is an enigmatic molecule, which was first synthesized in 1960 by the French anaesthesiologist H. Laborit in an attempt to generate a brain-permeable form of the principal inhibitory neurotransmitter g-aminobutyric acid (GABA) [1]. Three years later, Bessman and Fishbein reported GHB to be an endogenous constituent of the mammalian brain and a metabolite of GABA [2]. In 1982, Maitre and co-workers reported the existence of high-affinity [3H]GHB binding sites suggesting the existence of

Abbreviations: BnOPh-GHB, 4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate; DS, discriminative stimulus; GABA, g-aminobutyric acid; GABAB, GABA subtype B; GABAA, GABA subtype A; GBL, g-butyrolactone; GHB, g-hydroxybutyric acid; GPCR, G protein-coupled receptor; HOCPCA, 3-hydroxycyclopent-1-enecarboxylic acid; KO, knock-out; MAPK, mitogen-activated protein kinase; NCS-382, (2E)-(5hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid; NCS-400, 5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-yl ethanoic acid; NCS-435, g-(p-methoxybenzyl)-g-hydroxybutanoic acid; NCS-356, g-(p-chlorobenzyl)-g-hydroxybutanoic acid; PERV, porcine endogenous retrovirus; SSADH, succinic semialdehyde aldehyde dehydrogenase; SWD, spike-and-wave discharge; TM, transmembrane; T-HCA, trans-4-hydroxycrotonic acid; UMB68, 4-hydroxy-4methylpentanoic acid; UMB86, 4-hydroxy-4-naphtylbutanoic acid; WT, wild-type.. * Corresponding author. Tel.: +45 3917 9811; fax: +45 3530 6041. E-mail address: [email protected] (P. Wellendorph). 1 These authors have contributed equally. 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.10.028

specific GHB receptors [3]. GHB has attracted much public attention since the mid 1990s because of its recreational use. GHB was initially used by bodybuilders as an anabolic, but later it was widely abused as an illicit club drug (Fantasy or liquid ecstacy) for its euphoric, relaxing and sleep-promoting effects (for reviews see [4,5]). The latter effects have, regrettably, been exploited in situations of drug-facilitated sexual assault, but have also promoted GHB as a sleep-regulating clinically prescribed drug for treating symptoms of narcolepsy [6]. Other clinical uses of GHB include symptomatic treatment of alcohol withdrawal [7]. The many properties of GHB highlight its complex pharmacology, major aspects of which remain unclear. This review serves to give an update on GHB-specific pharmacology. This entails a description of the various reported receptors for GHB including the GABA subtype B (GABAB) receptors. We will particularly focus on the high-affinity binding sites for GHB and their possible functional roles and address the recent finding that parts of this population correspond to certain subtypes of GABA subtype A (GABAA) receptors. 1.1. GHB as a neurotransmitter Working alongside GABA in the neuronal synapse (Fig. 1), GHB fulfils relevant criteria for acting as a neurotransmitter or neuromodulator. GHB is synthesized from GABA in presynaptic neurons by a specific GHB synthesizing enzyme and can be

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Fig. 1. Overview of a GABA/GHBergic synapse. This overview shows the different low- and high-affinity targets for GHB, which are activated by either exogenous or synaptically released GHB. The metabotropic GABAB receptors and the ionotropic a4b2d and a4b3d GABAA receptors are activated by millimolar GHB concentrations only achieved after exogenous intake. The elusive GHB high-affinity sites, part of which may be represented by a4b1d GABAA receptors, are activated by GHB concentrations in the nanomolar to micromolar range. Abbreviations used: SSA, succinic semialdehyde; SSADH, SSA dehydrogenase; SSR, succinic semialdehyde reductase; SUC, succinate; GABA, g-aminobutyric acid; GABA-T, GABA transaminase; GHB, g-hydroxybutyric acid; GHB-T, GHB transporter; VIAAT, vesicular inhibitory amino acid transporter.

metabolized through the citric acid cycle via conversion into succinate by the enzyme succinic semialdehyde dehydrogenase (SSADH) [4]. Deficiency of SSADH is a pathological condition associated with increased levels of both GABA and GHB (also termed GHB aciduria) [8]. GHB is reported to be loaded into membrane vesicles via the vesicular inhibitory amino acid transporter, which also transports GABA and glycine. Via a Ca2+dependent mechanism, GHB is released into the synaptic cleft, where it may act on several different targets. GHB is removed from the synaptic cleft by a Na+-dependent plasma membrane transporter [9,10]. 2. GHB binding sites in the CNS In the CNS, GHB binds to both high- and low-affinity binding targets (Fig. 1). The most well-established target for GHB is the GABAB receptor, at which GHB displays weak affinity and is a partial agonist with millimolar potency [11,12]. However, endogenous GHB levels are in the micromolar range [10] and thereby several orders of magnitude too low to activate GABAB receptors. Consequently, GABAB receptor activation is most likely only observed after exogenous GHB intake, e.g. after therapeutic drug administration or recreational use [4,13]. Under such circumstances, GABAB receptors are highly relevant pharmacological targets for GHB and account for most of the reported in vivo effects of GHB as described in Section 3.

By contrast, the molecular identity of GHB high-affinity binding sites has remained elusive for decades. These binding sites, popularly referred to as elusive GHB receptors [14], are clearly distinct from GABAB receptors. Robust evidence for this was provided by binding studies in brains of GABAB1 knock-out (KO) mice, which display unchanged high-affinity GHB binding sites compared with wild-type (WT) littermates [15,16]. In parallel, successful generation of selective and high-affinity GHB analogues has prompted an interest in uncovering the molecular identity of the elusive GHB receptor and led to several candidate receptors being reported. In 2003, Maitre and co-workers reported the cloning of a GHB receptor from rat brain [17], and later in 2007, a human GHB receptor [18]. Interestingly, these two proteins do not appear to be orthologues and do not correlate with high-affinity binding site expression or pharmacology of GHB. In 2012, the GABAA receptor subunit a4 was shown to be involved in GHB highaffinity binding [19], and a4-containing GABAA receptor subtypes could represent a subpopulation of GHB receptors relevant under physiological circumstances [20]. 2.1. High-affinity binding sites 2.1.1. Localization and expression levels The [3H]GHB high-affinity binding sites were originally described by Maitre and co-workers as two populations of proteins of impressive expressional abundance (Kd1 = 30–580 nM,

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Bmax = 0.5–1.8 pmol/mg protein; and Kd2 = 2.3–16 mM, Bmax = 11– 46 pmol/mg protein) [10,13]. These binding sites appear to be conserved across species [3] and characterized by a distinct ontogenetic profile [21], which clearly differed from GABAB receptors [13]. [3H]GHB binding was specific, saturable and pHdependent with an optimum at pH 5.5 [3,21]. The regional expression of [3H]GHB high-affinity binding sites was initially investigated in membrane preparations from different rat brain regions [3]. High levels of [3H]GHB binding were detected in frontal cortex, olfactory bulbs, striatum and hippocampus and could not be displaced by GABA or by the selective GABAB receptor agonist, baclofen. Only low levels of binding were found in the cerebellum and peripheral organs [3]. This regional distribution of high-affinity binding sites in the rat brain was confirmed by quantitative autoradiography with the specific radioligand [3H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen6-ylidene) ethanoic acid ([3H]NCS-382) [22]. [3H]NCS-382 was found to label the same regional sites as [3H]GHB with the same pharmacological specificity, but without affinity for the GABAB receptor [22,23]. Therefore, [3H]NCS-382 has largely replaced [3H]GHB as the radioligand of choice for selective labelling of GHB high-affinity binding sites. More recently, the regional distribution and specificity of these sites were further confirmed using the GHB-specific [125I]BnOPh-GHB and [125I]azido-BnOPh-GHB radioligands (Fig. 2A) [24,25]. 2.1.2. Development of selective and high-affinity GHB tool compounds In addition to GABAB KO mice, ligands with selectivity for highaffinity GHB binding sites over GABAB receptors serve as promising tools to study isolated GHB effects. The first reported GHB analogue, trans-4-hydroxycrotonic acid (T-HCA) (Fig. 2B), was found to displace [3H]GHB binding with a slightly improved IC50 compared to GHB itself. Even higher affinity was achieved with the series of synthetic GHB analogues including NCS-382, g-(p-methoxybenzyl)-g-hydroxybutanoic acid (NCS435) and g-(p-chlorobenzyl)-g-hydroxybutanoic acid (NCS-356) (Fig. 2B) [26,27]. These compounds display high nanomolar affinity for the GHB binding site and no affinity for the GABAB receptor [16,22]. They have later been used as in vivo tool compounds with apparently opposite intrinsic activities; NCS-382 as a purported antagonist [27] and NCS-435/356 as agonists [28,29]. However, the antagonistic profile of NCS-382 appears to be somewhat ambiguous [14,30]. A saturated analogue of NCS-382, the compound 5-[4(aminoprop-1-ynyl)-phenyl]-4-hydroxypentanoic acid (NCS-400), was used for affinity-purification of the reported rat GHB receptor [17]. Using a strategy intended to limit potential metabolism of GHB analogues into GABA analogues [31], 4-substituted analogues such as the tertiary alcohol 4-hydroxy-4-methylpentanoic acid (UMB68) and 4-hydroxy-4-naphtylbutanoic acid (UMB86) containing a sterically hindered alcohol function, were developed and examined in vivo. These analogues represent interesting tool compounds but their affinities are not significantly improved compared to GHB. Two novel series of GHB analogues, represented by the conformationally restricted analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the 4-substituted biaromatic analogue 4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate (17b; BnOPh-GHB), markedly improved affinity compared with previous attempts (Fig. 2B) [32,33]. The racemic forms of HOCPCA and BnOPh-GHB showed 27 and 72 times higher affinity for the [3H]NCS-382 binding sites than GHB, respectively. Both were found to bind in stereospecific manners, which further improved affinity. Importantly, these compounds were devoid of affinity at GABAB ([3H]GABA) and GABAA ([3H]muscimol) binding sites [32,33]. HOCPCA and BnOPh-GHB are the compounds with the

Fig. 2. Regional distribution of the GHB high-affinity binding site and chemical structures of GHB and analogues (A) Autoradiograms showing the distribution of high-affinity GHB binding sites in rat brain using the specific GHB analogue [125I]BnOPh-GHB [24]. The binding is displaced by high concentrations of GHB, NCS-382, BnOPh-GHB but not GABA. The figure was reprinted from Wellendorph et al. [24] with permission from JPET. (B) Structural formulas of GHB and GABA analogues with affinity for [3H]NCS-382 binding sites. All structures are presented as racemic forms. Ki values represent inhibition of [3H]NCS-382 binding to rat brain cortical membranes and are collected from [19,32–34].

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highest affinity of the GHB analogues reported to date, and although their intrinsic activities are yet unknown, they constitute important tool compounds for studying effects mediated by GHB high-affinity sites [5]. The presence of the g-hydroxyl group has proven to be important to maintain high affinity, although exemptions have been reported. Interestingly, these included two GABA analogues: the non-steroid anti-inflammatory compound diclofenac and the GABAA receptor antagonist gabazine (SR95531) (Fig. 2B) [19,34]. The ability of gabazine to inhibit [3H]NCS-382 binding in an apparently competitive manner was unique among GABAA receptor ligands, which is intriguing considering that GHB is an agonist at certain GABAA receptors [19]. However, gabazine has also previously been reported to be an inhibitor of the monoamine oxidase A [35]. The high affinity of BnOPh-GHB led to subsequent characterization of the 125I-radiolabelled analogue [125I]BnOPh-GHB and the photoaffinity ligand [125I]azido-BnOPh-GHB [25]. [125I]BnOPhGHB labelled GHB binding sites in a regionally and pharmacologically specific manner using only picomolar concentrations (Kd of 7 nM) (Fig. 2A) [24], and [125I]azido-BnOPh-GHB facilitated the identification of novel high-affinity GHB targets as described in Section 4.1. 2.2. Functional relevance of the GHB high-affinity binding sites The existence of functionally relevant specific GHB receptormediated effects has been a matter of much investigation over the years. To firmly discriminate GHB effects at GABAB receptors from those mediated by high-affinity GHB binding sites, approaches that exclude GABAB receptor effects should be employed. These may include the use of (1) GABAB KO mice, (2) selective GABAB receptor antagonists and/or (3) specific GHB analogues without affinity for the GABAB receptor. 2.2.1. Role of GHB-specific effects in SSADH deficiency GHB is involved in the pathophysiology of SSADH deficiency, a clinical disorder associated with elevated GHB levels [8]. Therapeutic intervention has been limited to vigabatrin, an inhibitor of the enzyme g-aminobutyrate transaminase, which reduces levels of succinate semialdehyde and thereby GHB (Fig. 1), however, with limited clinical efficacy [8]. Gupta et al. investigated the effect of NCS-382 on the lifespan of SSADH-deficient mice (Aldh5a1 / ), which are neurologically impaired and die within four weeks after birth. Indeed, NCS-382 increased survival beyond 4 weeks by 50–61%, suggesting a direct role of the GHB receptor in the pathophysiology of SSADH deficiency [36]. 2.2.2. Effects of GHB on neuronal activity To link GHB high-affinity sites to function, neuronal activation in distinct brain regions has been studied. van Niewenhuijzen et al. assessed neuronal activation using c-Fos immunohistochemistry in rat brain after different doses of GHB sodium salt (250, 500 and 1000 mg/kg) and baclofen (10 mg/kg) [37]. Several brain regions, including the supraoptic and paraventricular nuclei of the hypothalamus and the central nucleus of the amygdala, were activated by both GHB and baclofen, indicating activation of GABAB receptors, GHB receptors or both. Interestingly, GHB but not baclofen induced c-Fos expression in the median raphe nucleus and the dentate gyrus of the hippocampus as well as in several brain regions involved in sleep physiology. Notably, the hippocampus is one of the regions with the highest expression of GHB high-affinity binding sites [3]. Altogether, these findings suggest that GHB exerts functional effects via its high-affinity site in a manner independent of the

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GABAB receptor. Studies using GABAB KO mice or specific GHB analogues would further substantiate these findings. 2.2.3. Effects of GHB on neurotransmitter release GHB was shown to modulate extracellular levels of several neurotransmitters, including GABA, glutamate and dopamine. Lower concentrations of GHB were reported to decrease GABA release via overlapping GABAB and GHB receptor-dependent mechanisms [29,38], whereas higher concentrations of GHB increased GABA release in a NCS-382-sensitive manner [29], leaving the exact involvement of GHB receptors unclear. Also, hippocampal glutamate levels were reported to be concentrationdependently regulated by GHB in vivo [28]. Intra-hippocampal (CA1) perfusion of nanomolar concentrations of GHB and the selective GHB analogues T-HCA and NCS-435 induced an NCS-382sensitive increase in glutamate levels that was not inhibited by the GABAB antagonist CGP35348. Conversely, a higher concentration of GHB (1 mM) inhibited glutamate release, which was counteracted by GABAB antagonists [28]. This convincingly suggests a role for GHB receptors at nanomolar concentrations of GHB as well as involvement of GABAB receptors at millimolar concentrations. Conversely, it was later reported that GHB (600 mM) decreased glutamate release by a mechanism sensitive to NCS-382, but resistant to the GABAB receptor antagonist CPG55845A [39]. It was proposed that the dual actions of GHB on neurotransmitter release were due to its preferential inhibition of GABAergic neurons as compared with dopaminergic neurons [40]. Thus, low doses of GHB would inhibit GABAergic neurons and indirectly allow increased firing of dopaminergic neurons. Higher doses of GHB would hyperpolarize both GABAergic and dopaminergic neurons. Future studies should seek to elucidate the underlying dual mechanisms of GHB, especially in the light of the recent finding that GHB can activate both GABAA and GABAB receptors. 2.2.4. Drug discrimination studies The receptors involved in the discriminative stimulus (DS) effect of GHB have been studied in vivo. DS studies illustrated that rats are able to discriminate GHB from water, and that such discriminating behaviour was fully [41] or partially blocked by NCS-382 [42]. The divergence in blocking efficiency by NCS-382 in these studies could be due to differences in doses, training regimes and animal species. Moreover, rats were able to discriminate GHB from baclofen [42], indicating that the DS properties of GHB could be mediated via stimulation of specific GHB receptors and not GABAB receptors. However, in the presence of high doses of NCS382 (100–200 mg/kg), DS properties of GHB were still present in the rats [43]. This may suggest that apart from being an antagonist at specific GHB receptors, NCS-382 could be an agonist under some conditions (for reviews see [30,44]). Interestingly, the high-affinity GHB analogue UMB86 could partly attenuate DS effects of GHB in rats trained to discriminate GHB from baclofen [31]. Also GABAB receptor antagonists attenuated the GHB-discriminating behaviour [43], indicating that both GABAB and GHB receptors play a role in GHB DS properties (for full review refer to Ref. [44]). 3. GABAB receptor-mediated effects of GHB 3.1. GHB as a GABAB receptor agonist The majority of reported pharmacological and behavioural effects of exogenous GHB are mediated via GABAB receptors. The GABAB receptor is a member of the family of G protein-coupled receptors characterized by the presence of a 7 transmembrane (TM) domain and a large extracellular amino-terminal ligandbinding domain [45]. GABAB receptor-mediated G protein

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activation leads to neuronal hyperpolarization by opening of postsynaptic G protein-coupled inwardly rectifying potassium channels and decreases in adenylate cyclase activity and calcium conductance. Indirect effects may also occur if GHB is converted to GABA, which in turn can activate GABAB (or GABAA) receptors. The order of magnitude of this process has not been entirely solved experimentally, but Ren & Mody showed that GHB-induced activation of GABAB receptors in vivo was not altered by inhibition of GHB conversion into GABA [46]. Likewise, binding to GABAB receptors in synaptosomes persisted in the presence of GHB dehydrogenase inhibitors [10]. The agonism of GHB at GABAB receptors was further confirmed using the GTPgS assay [15,28]. GHB (1 mM) gave rise to an increase in GTPgS, which was blocked by pre-treatment with the GABAB antagonist CGP35348, but not by the putative GHB receptor antagonist, NCS-382 [28]. These results were confirmed using GABAB KO mice [15]. Attempting to identify downstream effects of GHB-mediated GABAB receptor activation, Ren and Mody observed a long-lasting inhibition on mitogen-associated protein kinase (MAPK) phosphorylation in both cortex and hippocampus of mice after administration of GHB (500 mg/kg) and baclofen (20 mg/kg). This decrease was completely reversed by pre-treatment with the GABAB receptor antagonist CGP56999A [46]. Interestingly, GHB did not lead to decreased MAPK phosphorylation when cortical brain slices were studied in vitro following GHB administration. The same researchers later showed that administration of GHB also induced phosphorylation of cAMP response element-binding protein in the hippocampus [47]. However, when this experiment was preceded by chronic administration of GHB, no changes were observed, indicating that neuroadaptive changes had taken place. 3.2. Studies on GABAB1 receptor KO mice The GABAB1 KO mouse has been an excellent model to differentiate between GABAB and GHB receptor-mediated effects ([15,48–50], see Table 1). As mentioned earlier, [3H]GHB and [3H]NCS-382 binding was similar in GABAB1 KO mice and WT mice [15]. Notably, all pharmacological and behavioural effects (hypothermia, hypolocomotion, increase in striatal dopamine synthesis, EEG abnormalities) induced by GHB administration were absent in GABAB1 KO mice (Table 1), indicating a discrepancy between binding and functional studies. In line with the observations in GABAB1 KO mice, it was shown that the loss of righting reflex induced by 400 mg/kg g-butyrolactone (GBL) was completely reversed by pre-treatment with the GABAB receptor antagonist CGP35348 [51]. GBL is a commonly used prodrug of GHB with improved absorption and no affinity for GHB binding sites itself Table 1 Comparison of GHB-induced pharmacological effects in vivo. GHB-induced effects

Wild-type (WT) mice

GABAB1 knock-out (KO) mice Effect lost

Dopamine synthesis EEG delta waves EEG alpha, theta and beta2

Decreased (>300 mg/kg) Decreased (from 37 to 29 8C) Increased Increased Decreased

Effect lost (lower baseline temp) Effect lost Effect lost Effect lost

Que´va et al. [48] Core body temperature Behavioural score

Decreased Increased

Effect lost Effect lost

Vienne et al. [49] Locomotion EEG delta waves

Decreased Increased

Effect lost Effect lost

Kaupmann et al. [15] Total distance travelled Core body temperature

[3,52]. In summary, these studies demonstrated that all studied behavioural effects arising from GHB administration are mediated by the GABAB receptor, although compensatory mechanisms in the GABAB1 KO mice, presumably arising from phenotypic changes, such as memory impairment and suffering from epileptic seizures [48], cannot be ruled out. 3.3. Pharmacological significance of the GABAB receptor as a GHB target Exogenous administration of GHB induces euphoria, sedation, memory impairment, EEG abnormalities, dependence/abuse and ultimately coma and death in high doses [13]. Several studies observed a hypothermic effect of GHB [15,49], which was shown to be mediated by GABAB receptors. The GABAB receptor agonist baclofen is also known to lower the body temperature in mammals [49]. The observations that GHB lowered body temperature and cerebral glucose metabolism [53] makes it interesting in relation to neuroprotection. Despite the fact that GHB and baclofen both bind to and activate the GABAB receptor, important clinical differences have been observed. For example, no addiction properties of baclofen have been reported, and baclofen is not as effective as GHB in treating narcolepsy symptoms [44]. Complementary to the aforementioned GABAB1 KO mice studies, GHB pharmacology was also investigated in GHB-sensitive (GHB-S) and GHB-resistant (GHB-R) Wistar rat strains, which differ markedly in their response to both GHB and baclofen [54]. For example, GHB (1000 mg/kg) induced loss of the righting reflex in nearly all GHB-S rats, while it had no effect on the righting reflex in GHB-R rats, thus GHB-R rats have traits similar to GABAB1 KO mice [15]. The same was evident using baclofen [41]. Furthermore, it was observed that GHB and baclofen administration showed higher induction of the endogenous neurosteroids 3a,5a-THP and 3a,5a-THDOC in GHB-S rats compared with GHB-R rats [54]. Also, when pregnanolone was pre-administered to GHB-R rats, GHBinduced loss of the righting reflex was observed in several of the GHB-R rats, and the duration of sedation was prolonged [54]. This suggests that endogenous neurosteroids may modulate the GHB sedative-hypnotic effect. 4. Going from B to A: GABAA receptor subtypes as novel GHB receptors 4.1. Identification of a4bd GABAA receptors as GHB high-affinity targets GABAA receptors are heteropentameric ligand-gated ion channels, which induce hyperpolarization by increasing chloride conductance. A wide variety of subtypes can be formed from the 19 known subunits, and pharmacological properties, expression pattern and location in the synapse are determined by subunit composition [55]. We recently presented molecular evidence that a certain subpopulation of GABAA receptors are high-affinity GHB receptors [19]. A GHB high-affinity binding protein was isolated using the selective photosensitive radioligand [125I]azido-BnOPhGHB [25], and high-resolution mass spectrometric analysis identified several GABAA receptor subunits within the isolated samples. Systematic testing at human recombinant GABAA receptor subtypes expressed in X. laevis oocytes revealed that GHB was a partial agonist at a4bd receptors with a relative agonist efficacy of 53–76% of maximum GABA response (Fig. 3A and B). GHB was particularly potent at a4b1d receptors (EC50 = 140 nM) as compared with a4b2d and a4b3d, which had EC50 values in the low millimolar range. Only very small GHB responses, if any, were observed in a(1,2,6)b1d, ab and abg2

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Fig. 3. Pharmacological characterization of GHB at recombinant a4bd GABAA receptors expressed in Xenopus laevis oocytes (A, B, D) and [3H]NCS-382 binding sites in a4 KO mice (C). (A) Representative GHB current traces at a4b1d GABAA receptors. (B) Concentration-response curve of GHB at a4b(1–3)d receptors normalized to maximal GABA response (means  SEM, n = 4–6). (C) Saturation of [3H]NCS-382 binding to membrane preparations from a4 knock-out (KO) mice and wild-type (WT) littermates. The number of binding sites was significantly reduced in a4 KO mice (P < 0.01, Student’s t-test, n = 5). Graphs were mathematically converted from homologous binding competition data. (D) Abolishment of GHB response by a4F71L point mutation in a4F71Lb1 and a4F71Lb1d receptors as compared with WT receptors. Bar diagrams show mean effects  SEM of 30 mM GHB at a4b1 vs. a4F71Lb1 receptors and 100 mM GHB at a4b1d vs. a4F71Lb1d receptors (***P < 0.001 and *P < 0.05, Student’s t-test). (E) Left, structural overview of the pentameric chloride-conducting a4b1d GABAA receptor. Right, the a4b1d receptor viewed from the extracellular side. The location of the GABA binding sites (white triangles) and the a4F71L point mutations are shown. Subpanels A–D were reprinted from Absalom et al. [19] with permission from PNAS.

subtypes. Overall, GHB was dependent on a4 and d subunits for efficacy and on b1 for potency. To link this finding to the native GHB high-affinity site, we investigated [3H]NCS-382 binding in a4 and d KO mice. [3H]NCS-382 binding was unchanged in d KO mice, indicating that although d is essential for the GHB response, it is not necessary for GHB binding. However, the number of GHB highaffinity binding sites was reduced by 39% in a4 KO mice brains compared with WT littermates, suggesting that a4 is involved in GHB high-affinity binding (Fig. 3C). Although the regional distribution of GHB high-affinity binding sites does not clearly match that of a4, the a4 subunit is expressed widely in forebrain regions. Intriguingly, the distribution of b1 overlaps much more convincingly with the GHB binding sites (Fig. 2A) [56]. The GHB response in a4b1d was completely blocked by the orthosteric GABAA receptor antagonist gabazine and by the point mutation a4F71L (Fig. 3D and E), identifying a4F71 as a key residue in mediating GHB activity. The a4F71L point mutation also reduced GABA EC50 5-fold, indicating that the GHB binding site is overlapping, but not identical to the GABA and gabazine binding sites in the ba interface. The homologous point mutation in the a1 subunit (a1F64L) drastically reduced GABA EC50 and the IC50 values of the competitive antagonists bicuculline and gabazine at a1b2g2 receptors, indicating that this mutation directly alters the orthosteric agonist/antagonist binding site [57]. In summary, it was proposed that a4bd receptors constitute a GHB high-affinity target with the binding site located in the extracellular ba interface, where it overlaps with the GABA and gabazine binding sites in a mutually non-exclusive manner. The remaining [3H]NCS-382 binding in a4 KO mice (61%) are proposed to represent other populations of highaffinity sites, possibly alternative GABAA receptor ba interfaces [19]. 4.2. GHB as a GABAA receptor agonist GHB is structurally very similar to GABA and shares its pharmacological properties with sedative-hypnotic compounds that act at the GABAA receptor, and it might therefore seem likely

for GHB to interact with the GABAAergic system. However, this matter has been a subject of debate for several decades. In 1987, Snead and Nichols reported that [3H]GHB binding was enhanced by pentobarbital, picrotoxin and diazepam, which all modulate the GABA/benzodiazepine/picrotoxin complex, leading to the hypothesis that the GHB binding site was coupled to the GABAA receptor [58]. By contrast, radioligand binding studies have consistently shown that GHB high-affinity binding cannot be modulated by GABA or GABA agonists [3,19,22], and that GHB does not directly affect the GABAA receptor in terms of [3H]flunitrazepam and [3H]muscimol binding or muscimol-induced 36Cl uptake in rat cortex [59,60]. Whereas GHB and GABA were both shown to hyperpolarize cultured rat spinal neurons by increasing chloride conductance in a bicuculline-sensitive manner, suggesting a GABA-mimicking effect of GHB, possibly by a direct GABAA receptor activation [61], other studies have found only GABAB-mediated effects [62]. GHB was also shown to reduce firing in nigral and neocortical neurons via a bicuculline-insensitive mechanism [63]. Furthermore, Osorio and Davidoff observed opposite polarizing effects of GHB and GABA in frog dorsal root terminals, where only the GABA response was inhibited by picrotoxin and bicuculline, indicating that GHB is not a GABA agonist [64]. Yet another study reported NCS-382-sensitive inhibition of a GABAA receptor-mediated inhibitory postsynaptic potential by GHB in the presence of a GABAB receptor antagonist in CA1 hippocampal neurons [65]. More recent studies suggest a role for GHB at extrasynaptic GABAA receptors, specifically subtypes containing the a4 and d subunits. A connection between GHB and d was indicated by a study of the well-characterized model of epileptic generalized absence seizures, which are induced by GHB and characterized by spike-and-wave discharges (SWDs). GHB-induced SWDs were absent in d KO mice and in rats after knock-down of d subunit expression. Furthermore, the d-preferring GABAA receptor agonist THIP induced SWDs and absence seizures in naı¨ve rats [66], indicating similar neuropharmacological effects of THIP and GHB.

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A metabolomics study showed that the metabolic fingerprint of GHB (1 mM) in guinea pig cortex was similar to that of GABA, the d-preferring agonist THIP and a subset of GABAA allosteric modulators acting at a 4-containing receptors [67]. Most recently, Thompson et al. showed that GHB was a weak partial agonist at the Erwinia ligand-gated ion channel, a bacterial homologue of vertebrate Cys-loop receptors, which is activated by GABA and shares many structural features with GABAA receptors [68]. The abovementioned electrophysiological studies arguing against GABAA receptor involvement in GHB pharmacology were all conducted in native tissue. In contrary to the GABAB receptor, a broad variety of GABAA receptor subtypes exist, all exhibiting distinct pharmacological properties and yet unknown patterns of regional expression [55]. According to our previous findings [19], only GHB activation of the particular subtype a4b1d (and, at millimolar GHB concentrations, a4b 2d and a4b 3d) would be of pharmacological relevance, which complicates investigation of GHB physiology and pharmacology in native tissue. This could be one explanation for the lack of observed in vitro GABAA effects and responses to submillimolar GHB concentrations in the above-mentioned electrophysiological studies in native tissue. 4.3. Physiological and pharmacological significance of GHB-mediated activation of the a4b1d receptor The a4b1d receptor subtype was identified as an important target for endogenous neurosteroids, and its expression was modulated by fluctuating hormone levels throughout the oestrous cycle [69]. Besides cortex and hippocampus, a4, b1 and d subunits are co-expressed in brain regions important for modulation of sleep and wakefulness as well as facilitation of EEG synchronization such as the reticular thalamic nucleus [70,71]. The therapeutic effect of GHB in narcolepsy may thus in part be explained by its preference for the b1 subunit, supported by the report that b1containing receptors were involved in mediation of sleep, whereas b3-containing receptors mediated hypnosis and anaesthesia [72]. Furthermore, a correlation was shown between the activity at b1containing receptors and ataxia [71], which is interesting since ataxia was previously proposed to be a GHB-specific effect [73]. The physiological relevance of the agonistic effect of GHB at recombinant a4bd receptors is yet to be clarified. Most likely, GHB has multiple mechanisms of action, possibly involving direct GABAA receptor activation or indirect activation via GABAB receptor activation and/or increased levels of endogenous neurosteroids [54]. In support of this, recent studies demonstrated a functionally relevant postsynaptic crosstalk between GABAB and dcontaining GABAA receptors [74]. It was shown that GHB enhanced tonic inhibition, presumably at extrasynaptic a4b1d receptors, via GABAB receptor activation by a pathway dependent on Gi/o proteins, adenylate cyclase and cAMP-dependent protein kinase A [74]. Clearly, complex interactions between GHB, GABAB and GABAA receptors exist, and elaborate studies are needed to clarify these. 5. Other reported GHB receptors In 2003, Maitre and co-workers reported the cloning of a novel GHB receptor from rat brain [17]. Using the analogue NCS400 (Fig. 2B), a protein from solubilized rat brain membranes was isolated by affinity purification. The resulting protein of 56 kDa corresponds well with the size of the protein reported using a similar approach and the GHB-derived photoaffinity ligand [125I]azido-BnOPh-GHB [24]. Several peptides were isolated and used to design degenerated PCR primers, which

led to the isolation of a putative rat GHB receptor [17]. Sequence analysis showed that the cloned protein display homology to a class of tetraspanin proteins with a characteristic 4TM domain, nonetheless the authors classified the protein as a G proteincoupled receptor. Pharmacological characterization showed some traits reminiscent of a GHB receptor, i.e. [3H]GHB binding and an electrophysiological response to GHB, although this was not antagonized by NCS-382. Thus, several points argue against the idea that this receptor protein corresponds to the [3H]NCS382 high-affinity binding site, as both expressional and pharmacological profiles deviate [5,14]. Later, the same group reported cloning of a human GHB receptor. This protein showed only 50–60% sequence identity and no apparent homology with the previously reported rat receptor, although it was cloned based on the reported rat receptor cDNA sequence [18]. The isolated putative human GHB receptor was named GHBh1 and was shown to bind [3H]GHB in heterologous systems. As briefly mentioned in the paper, the isolated protein is also known as GPR41/GPR172A. Closer examination of the protein sequence reveals that additional names and functions exist for this apparent GHB receptor. Previously referred to as GPR172A or PERV-A receptor (HuPAR-1), this protein was identified as a receptor for porcine endogenous retroviruses (PERV) and shown to be highly expressed in testis and very little in the CNS [75]. A more recent report states that the protein is in fact a riboflavin transporter and should thus correctly be referred to as RFT2 (GenBank accession no. NM_024531.4) [76]. Thus, the reported human GHB receptor sequence isolated by Andriamampandry et al. is identical to RFT2; a protein with a 10TM domain which transports riboflavin under normal conditions and which recognizes viruses under acquired conditions. In conclusion, the reported GHB receptors do not seem to be 7TM GPCRs, nor do they fulfil criteria that fit with [3H]NCS-382 high-affinity binding sites, but they may be GHB-interacting proteins with other pharmacological roles.

6. Conclusion and perspectives Although GHB and its high-affinity binding sites have been known for several decades, their precise role in the mammalian CNS remains largely unknown. This review has attempted to provide a balanced research update on the different reported receptor targets for GHB, and their proposed pharmacological and physiological roles. GABAB receptors are low-affinity targets of prime pharmacological importance in relation to high-dose GHB effects, but cannot explain high-affinity binding and effects. This has been corroborated through studies using GABAB KO mice. The recent identification of a4bd GABAA receptors as highaffinity GHB targets has highlighted a potential role for GHB at extrasynaptic sites and thus provided a new exciting dimension for GHB neuropharmacology. How does GHB interact with GABAA receptors on the molecular level? And what is the physiological relevance of this interaction? To begin to address such questions, electrophysiological recordings in GABAA KO brain tissue or neuronal cells constitute important starting points, which may be supplemented with in vivo studies using selective GHB analogues. Still only a subset of GHB high-affinity sites have been identified (40%). This leaves the full understanding of GHB pharmacology and function an enigmatic subject for future research. Acknowledgements This work was supported by the Carlsberg Foundation, the Lundbeck Foundation and the Drug Research Academy.

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References [1] Laborit H. Sodium 4-Hydroxybutyrate. Int J Neuropharm 1964;3:433–51. [2] Bessman SP, Fishbein WN. g-Hydroxybutyrate, a normal brain metabolite. Nature 1963;200:1207–8. [3] Benavides J, Rumigny JF, Bourguignon JJ, Cash C, Wermuth CG, Mandel P, et al. High affinity binding sites for g-hydroxybutyric acid in rat brain. Life Sci 1982;30:953–61. [4] Drasbek KR, Christensen J, Jensen K. Gamma-hydroxybutyrate—a drug of abuse. Acta Neurol Scand 2006;114:145–56. [5] Castelli MP. Multi-faceted aspects of g-hydroxybutyric acid: a neurotransmitter, therapeutic agent and drug of abuse. Mini Rev Med Chem 2008;8:1188– 202. [6] Tunnicliff G, Raess BU. g-Hydroxybutyrate (orphan medical). Curr Opin Investig Drugs 2002;3:278–83. [7] Addolorato G, Leggio L, Ferrulli A, Caputo F, Gasbarrini A. The therapeutic potential of g-hydroxybutyric acid for alcohol dependence: balancing the risks and benefits. A focus on clinical data. Expert Opin Investig Drugs 2009;18:675– 86. [8] Gibson KM, Hoffmann GF, Hodson AK, Bottiglieri T, Jakobs C. 4-Hydroxybutyric acid and the clinical phenotype of succinic semialdehyde dehydrogenase deficiency, an inborn error of GABA metabolism. Neuropediatrics 1998;29:14–22. [9] Benavides J, Rumigny JF, Bourguignon JJ, Wermuth CG, Mandel P, Maitre M. A high-affinity, Na+-dependent uptake system for g-hydroxybutyrate in membrane vesicles prepared from rat brain. J Neurochem 1982;38:1570–5. [10] Maitre M. The g-Hydroxybutyrate signalling system in brain: organization and functional implications. Prog Neurobiol 1997;51:337–61. [11] Mathivet P, Bernasconi R, De Barry J, Marescaux C, Bittiger H. Binding characteristics of g-hydroxybutyric acid as a weak but selective GABAB receptor agonist. Eur J Pharmacol 1997;321:67–75. [12] Lingenhoehl K, Brom R, Heid J, Beck P, Froestl W, Kaupmann K, et al. gHydroxybutyrate is a weak agonist at recombinant GABAB receptors. Neuropharmacology 1999;38:1667–73. [13] Bernasconi R, Mathivet P, Bischoff S, Marescaux C. Gamma-hydroxybutyric acid: an endogenous neuromodulator with abuse potential. Trends Pharmacol Sci 1999;20:135–41. [14] Crunelli V, Emri Z, Leresche N. Unravelling the brain targets of g-hydroxybutyric acid. Curr Opin Pharmacol 2006;6:44–52. [15] Kaupmann K, Cryan JF, Wellendorph P, Mombereau C, Sansig G, Klebs K, et al. Specific g-hydroxybutyrate-binding sites but loss of pharmacological effects of g-hydroxybutyrate in GABAB(1)-deficient mice. Eur J Neurosci 2003;18:2722–30. [16] Wu Y, Ali S, Ahmadian G, Liu CC, Wang YT, Gibson KM, et al. g-Hydroxybutyric acid (GHB) and g-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence. Neuropharmacology 2004;47:1146–56. [17] Andriamampandry C, Taleb O, Viry S, Muller C, Humbert JP, Gobaille S, et al. Cloning and characterization of a rat brain receptor that binds the endogenous neuromodulator g-hydroxybutyrate (GHB). Faseb J 2003;17:1691–3. [18] Andriamampandry C, Taleb O, Kemmel V, Humbert JP, Aunis D, Maitre M. Cloning and functional characterization of a g-hydroxybutyrate receptor identified in the human brain. Faseb J 2007;21:885–95. [19] Absalom N, Eghorn LF, Villumsen IS, Karim N, Bay T, Olsen JV, et al. a4bd GABAA receptors are high-affinity targets for g-hydroxybutyric acid (GHB). Proc Natl Acad Sci USA 2012;109:13404–09. [20] Enna SJ. Extrasynaptic site of action for g-hydroxybutyrate. Proc Natl Acad Sci USA 2012;109:13142–43. [21] Snead 3rd OC. The ontogeny of [3H]g-hydroxybutyrate and [3H]GABAB binding sites: relation to the development of experimental absence seizures. Brain Res 1994;659:147–56. [22] Mehta AK, Muschaweck NM, Maeda DY, Coop A, Ticku MK. Binding characteristics of the g-hydroxybutyric acid receptor antagonist [3H](2E)-(5-hydroxy5,7,8,9-tetrahydro-6H-benzo[a][7] annulen-6-ylidene) ethanoic acid in the rat brain. J Pharmacol Exp Ther 2001;299:1148–53. [23] Gould GG, Mehta AK, Frazer A, Ticku MK. Quantitative autoradiographic analysis of the new radioligand [3H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6Hbenzo[a][7] annulen-6-ylidene) ethanoic acid ([3H]NCS-382) at g-hydroxybutyric acid (GHB) binding sites in rat brain. Brain Res 2003;979:51–6. [24] Wellendorph P, Høg S, Sabbatini P, Pedersen MH, Martiny L, Knudsen GM, et al. Novel radioiodinated GHB analogues for radiolabeling and photolinking of high-affinity GHB binding sites. J Pharmacol Exp Ther 2010;335:458–64. [25] Sabbatini P, Wellendorph P, Høg S, Pedersen MHF, Bra¨uner-Osborne H, Martiny L, et al. Design, synthesis and in vitro pharmacology of new radiolabelled GHB analogues including photolabile analogues with irreversible binding to the high-affinity GHB binding sites. J Med Chem 2010;53:6506–10. [26] Bourguignon JJ, Schmitt M, Didier B. Design and structure–activity relationship analysis of ligands of g-hydroxybutyric acid receptors. Alcohol 2000;20:227–36. [27] Maitre M, Hechler V, Vayer P, Gobaille S, Cash CD, Schmitt M, et al. A specific ghydroxybutyrate receptor ligand possesses both antagonistic and anticonvulsant properties. J Pharmacol Exp Ther 1990;255:657–63. [28] Castelli MP, Ferraro L, Mocci I, Carta F, Carai MA, Antonelli T, et al. Selective ghydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/ hypnotic effect of g-hydroxybutyric acid. J Neurochem 2003;87:722–32.

227

[29] Gobaille S, Hechler V, Andriamampandry C, Kemmel V, Maitre M. g-Hydroxybutyrate modulates synthesis and extracellular concentration of g-aminobutyric acid in discrete rat brain regions in vivo. J Pharmacol Exp Ther 1999;290:303–9. [30] Castelli MP, Pibiri F, Carboni G, Piras AP. A review of pharmacology of NCS-382, a putative antagonist of g-hydroxybutyric acid (GHB) receptor. CNS Drug Rev 2004;10:243–60. [31] Carter LP, Wu H, Chen W, Matthews MM, Mehta AK, Hernandez RJ, et al. Novel g-hydroxybutyric acid (GHB) analogs share some, but not all, of the behavioral effects of GHB and GABAB receptor agonists. J Pharmacol Exp Ther 2005;313:1314–23. [32] Wellendorph P, Høg S, Greenwood JR, de Lichtenberg A, Nielsen B, Frølund B, et al. Novel cyclic g-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain. J Pharmacol Exp Ther 2005;315:346–51. [33] Høg S, Wellendorph P, Nielsen B, Frydenvang K, Dahl IF, Bra¨uner-Osborne H, et al. Novel high-affinity and selective biaromatic 4-substituted g-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies. J Med Chem 2008;51:8088–95. [34] Wellendorph P, Høg S, Skonberg C, Bra¨uner-Osborne H. Phenylacetic acids and the structurally related non-steroidal anti-inflammatory drug (NSAID) diclofenac bind to specific g-hydroxybutyric acid (GHB) sites in rat brain. Fund Clin Pharmacol 2009;23:207–13. [35] Luque JM, Erat R, Kettler R, Cesura A, Da Prada M, Richards JG. Radioautographic evidence that the GABAA receptor antagonist SR 95531 is a substrate inhibitor of MAO-A in the rat and human locus coeruleus. Eur J Neurosci 1994;6:1038–49. [36] Gupta M, Greven R, Jansen EE, Jakobs C, Hogema BM, Froestl W, et al. Therapeutic intervention in mice deficient for succinate semialdehyde dehydrogenase (g-hydroxybutyric aciduria). J Pharmacol Exp Ther 2002;302:180– 7. [37] van Nieuwenhuijzen PS, McGregor IS, Hunt GE. The distribution of g-hydroxybutyrate-induced Fos expression in rat brain: comparison with baclofen. Neuroscience 2009;158:441–55. [38] Hu RQ, Banerjee PK, Snead 3rd OC. Regulation of g-aminobutyric acid (GABA) release in cerebral cortex in the g-hydroxybutyric acid (GHB) model of absence seizures in rat. Neuropharmacology 2000;39:427–39. [39] Brancucci A, Berretta N, Mercuri NB, Francesconi W. Gamma-hydroxybutyrate and ethanol depress spontaneous excitatory postsynaptic currents in dopaminergic neurons of the Substantia nigra. Brain Res 2004;997:62–6. [40] Cruz HG, Ivanova T, Lunn ML, Stoffel M, Slesinger PA, Luscher C. Bi-directional effects of GABAB receptor agonists on the mesolimbic dopamine system. Nat Neurosci 2004;7:153–9. [41] Colombo G, Agabio R, Balaklievskaia N, Diaz G, Lobina C, Reali R, et al. Oral selfadministration of g-hydroxybutyric acid in the rat. Eur J Pharmacol 1995;285:103–7. [42] Koek W, Chen W, Mercer SL, Coop A, France CP. Discriminative stimulus effects of g-hydroxybutyrate: role of training dose. J Pharmacol Exp Ther 2006;317:409–17. [43] Carter LP, Flores LR, Wu H, Chen W, Unzeitig AW, Coop A, et al. The role of GABAB receptors in the discriminative stimulus effects of g-hydroxybutyrate in rats: time course and antagonism studies. J Pharmacol Exp Ther 2003;305:668–74. [44] Carter LP, Koek W, France CP. Behavioral analyses of GHB: receptor mechanisms. Pharmacol Ther 2009;121:100–14. [45] Bettler B, Kaupmann K, Mosbacher J, Gassmann M. Molecular structure and physiological functions of GABAB receptors. Physiol Rev 2004;84:835–67. [46] Ren X, Mody I. g-Hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABAB receptor activation in mouse frontal cortex and hippocampus. J Biol Chem 2003;278:42006–11. [47] Ren X, Mody I. g-Hydroxybutyrate induces cyclic AMP-responsive elementbinding protein phosphorylation in mouse hippocampus: an involvement of GABAB receptors and cAMP-dependent protein kinase activation. Neuroscience 2006;141:269–75. [48] Schuler V, Lu¨scher C, Blanchet C, Klix N, Sansig G, Klebs K, et al. Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABAB responses in mice lacking GABAB(1). Neuron 2001;31:47–58. [49] Que´va C, Bremner-Danielsen M, Edlund A, Ekstrand AJ, Elg S, Erickson S, et al. Effects of GABA agonists on body temperature regulation in GABAB(1) / mice. Br J Pharmacol 2003;140:315–22. [50] Vienne J, Bettler B, Franken P, Tafti M. Differential effects of GABAB receptor subtypes, g-hydroxybutyric acid, and baclofen on EEG activity and sleep regulation. J Neurosci 2010;30:14194–204. [51] Carai MAM, Lobina C, Maccioni P, Cabras C, Colombo G, Gessa GL. g-Aminobutyric acidB (GABAB)-receptor mediation of different in vivo effects of gbutyrolactone. J Pharmacol Sci 2008;106:199–207. [52] Roth RH, Delgado JM, Gamma-butyrolactone Giarman NJ. gamma-hydroxybutyric acid. II. The pharmacologically active form. Int J Neuropharmacol 1966;5:421–8. [53] Kuschinsky W, Suda S, Sokoloff L. Influence of g-hydroxybutyrate on the relationship between local cerebral glucose utilization and local cerebral blood flow in the rat brain. J Cereb Blood Flow Metab 1985;5:58–64. [54] Barbaccia ML, Carai MA, Colombo G, Lobina C, Purdy RH, Gessa GL. Endogenous g-aminobutyric acid GABAA receptor active neurosteroids and the sedative/ hypnotic action of g-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines. Neuropharmacology 2005;49:48–58.

228

T. Bay et al. / Biochemical Pharmacology 87 (2014) 220–228

[55] Olsen RW, Sieghart W. GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 2009;56:141–8. [56] Ho¨rtnagl H, Tasan RO, Wieselthaler A, Kirchmair E, Sieghart W, Sperk G. Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain. Neuroscience 2013;236:345–72. [57] Sigel E, Baur R, Kellenberger S, Malherbe P. Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels. EMBO J 1992;11:2017–23. [58] Snead OCI, Nichols AC. g-Hydroxybutyric acid binding sites: evidence for coupling to a chloride anion channel. Neuropharmacology 1987;26:1519–23. [59] Serra M, Sanna E, Foddi C, Concas A, Biggio G. Failure of g-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex. Psychopharmacol (Berl) 1991;104:351–5. [60] Snead OC. 3rd, Liu CC. GABAA receptor function in the g-hydroxybutyrate model of generalized absence seizures. Neuropharmacology 1993;32:401–9. [61] Ho¨sli L, Ho¨sli E, Lehmann R, Schneider J, Borner M. Action of g-hydroxybutyrate and GABA on neurones of cultured rat central nervous system. Neurosci Lett 1983;37:257–60. [62] Xie X, Smart TG. Gamma-hydroxybutyrate hyperpolarizes hippocampal neurones by activating GABAB receptors. Eur J Pharmacol 1992;212:291–4. [63] Olpe HR, Koella WP. Inhibition of nigral and neocortical cells by g-hydroxybutyrate: a microiontophoretic investigation. Eur J Pharmacol 1979;53:359–64. [64] Osorio I, Davidoff RA. g-Hydroxybutyric acid is not a GABA-mimetic agent in the spinal cord. Ann Neurol 1979;6:111–6. [65] Cammalleri M, Brancucci A, Berton F, Loche A, Gessa GL, Francesconi W. Gamma-hydroxybutyrate reduces GABAA-mediated inhibitory postsynaptic potentials in the CA1 region of hippocampus. Neuropsychopharmacol 2002;27:960–9. [66] Cope DW, Di Giovanni G, Fyson SJ, Orban G, Errington AC, Lorincz ML, et al. Enhanced tonic GABAA inhibition in typical absence epilepsy. Nat Med 2009;15:1392–8.

[67] Nasrallah FA, Maher AD, Hanrahan JR, Balcar VJ, Rae CD. g-Hydroxybutyrate and the GABAergic footprint: a metabolomic approach to unpicking the actions of GHB. J Neurochem 2010;115:58–67. [68] Thompson AJ, Alqazzaz M, Ulens C, Lummis SC. The pharmacological profile of ELIC, a prokaryotic GABA-gated receptor. Neuropharmacology 2012;63: 761–7. [69] Lovick TA, Griffiths JL, Dunn SM, Martin IL. Changes in GABAA receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats. Neuroscience 2005;131:397–405. [70] Sergeeva OA, Kletke O, Kragler A, Poppek A, Fleischer W, Schubring SR, et al. Fragrant dioxane derivatives identify b1-subunit-containing GABAA receptors. J Biol Chem 2010;285:23985–93. [71] Gee KW, Tran MB, Hogenkamp DJ, Johnstone TB, Bagnera RE, Yoshimura RF, et al. Limiting activity at b1-subunit-containing GABAA receptor subtypes reduces ataxia. J Pharm Exp Ther 2010;332:1040–53. [72] Yanovsky Y, Schubring S, Fleischer W, Gisselmann G, Zhu XR, Lubbert H, et al. GABAA receptors involved in sleep and anaesthesia: b1- versus b3-containing assemblies. Pflugers Archiv Eur J Phys 2012;463:187–99. [73] Carter LP, Chen W, Wu H, Mehta AK, Hernandez RJ, Ticku MK, et al. Comparison of the behavioral effects of g-hydroxybutyric acid (GHB) and its 4-methylsubstituted analog, g-hydroxyvaleric acid (GHV). Drug Alcohol Depend 2005;78:91–9. [74] Connelly WM, Fyson SJ, Errington AC, McCafferty CP, Cope DW, Di Giovanni G, et al. GABAB receptors regulate extrasynaptic GABAA receptors. J Neurosci 2013;33:3780–5. [75] Ericsson TA, Takeuchi Y, Templin C, Quinn G, Farhadian SF, Wood JC, et al. Identification of receptors for pig endogenous retrovirus. Proc Natl Acad Sci USA 2003;100:6759–64. [76] Yao Y, Yonezawa A, Yoshimatsu H, Masuda S, Katsura T, Inui K. Identification and comparative functional characterization of a new human riboflavin transporter hRFT3 expressed in the brain. J Nutr 2010;140:1220–6.