In vivo labelling of α5 subunit-containing GABAA receptors using the selective radioligand [3H]L-655,708
Introduction
Mammalian GABAA receptors are generally accepted to be pentameric assemblies of subunits derived from the 19 members of the GABAA receptor subunit family (α1–6, β1–3, γ1–3, δ, ɛ, θ, π and ρ1–3) (Barnard et al., 1998, Bonnert et al., 1999) with the majority of native receptors containing α, β and γ subunits in a 2:2:1 stoichiometry (Sieghart and Sperk, 2002). In addition to playing a major role in mediating the effects of GABA, the main inhibitory neurotransmitter within the CNS, the GABAA receptor also contains a number of modulatory sites that allosterically modify receptor function and are thought to be responsible for the pharmacological and clinical effects of a diverse range of compounds including barbiturates, certain anaesthetics, neurosteroids and benzodiazepines (Sieghart, 1995, Korpi et al., 2002).
The pharmacology of GABAA receptors that possess a benzodiazepine binding site is dictated by the α and γ subunits contained within the receptor, since the binding site occurs at the interface of these two subunits (McKernan and Whiting, 1996). Furthermore, since the major γ subunit occurring in native receptors is γ2, heterogeneity in benzodiazepine pharmacology of GABAA receptors in the brain is primarily a function of the α subunit present (McKernan and Whiting, 1996). For example, non-selective benzodiazepines, such as diazepam, have equivalent affinity for the benzodiazepine binding site of GABAA receptors containing an α1, α2, α3 or α5 subunit yet have essentially no affinity for GABAA receptors containing an α4 or α6 subunit; a selectivity solely attributable to the presence of an arginine residue in the α4 and α6 subunit which replaces a histidine residue found in α1, α2, α3 and α5 (Wieland et al., 1992, Benson et al., 1998).
Non-selective benzodiazepines, such as diazepam, possess a number of behavioural properties (e.g. anxiolysis, sedation and anticonvulsant activity) and the heterogeneous distribution of mRNA and protein for the different α subunits within the brain suggests that there may be functional heterogeneity of benzodiazepine modulatory actions within the CNS (Wisden et al., 1992, Fritschy and Mohler, 1995, Pirker et al., 2000). In other words, particular behavioural properties of benzodiazepines may be associated with specific α subunit-containing subtypes of the GABAA receptor (Lüddens et al., 1995). Recently, transgenic mice in which particular α subunits have been rendered insensitive to diazepam by changing the histidine residue which is crucial for diazepam binding to an arginine (Benson et al., 1998) have been used to help delineate which GABAA receptor subtypes are associated with which of the pharmacological properties of diazepam (Rosahl, 2003, Rudolph and Möhler, 2004). For example, α1-containing GABAA receptors are involved in benzodiazepine-induced sedation (Rudolph et al., 1999, McKernan et al., 2000), a role confirmed pharmacologically by the fact that L-838417, a compound which does not modulate the function of α1-containing receptors, demonstrates reduced sedative liability (McKernan et al., 2000) and zolpidem, which interacts relatively selectively with α1 subunit-containing GABAA receptors is hypnotic (Rush, 1998).
L-655,708 is an imidazobenzodiazepine which possesses greater affinity for α5 versus α1, α2 or α3 subunit-containing GABAA receptors (Quirk et al., 1996). [3H]L-655,708 has been used to show autoradiographically that α5 subunit-containing GABAA receptors have a high level of expression in the rat and human hippocampus (Sur et al., 1999, Howell et al., 2000, Li et al., 2001), in agreement with in situ hybridization and immunohistochemical studies (Wisden et al., 1992, Fritschy and Mohler, 1995, Pirker et al., 2000). The function of α5-containing GABAA receptors is not well understood, although their relatively high expression in the hippocampus implicates this particular subtype in hippocampal-dependent processes such as cognition, a role supported by recent evidence from α5 knock-out and diazepam-insensitive transgenic mice (Collinson et al., 2002, Crestani et al., 2002) as well as compounds with α5 selectivity (Chambers et al., 2002, Chambers et al., 2003, Sternfeld et al., 2004). More recently, this GABAA receptor subtype has also been implicated in the mechanism of pre-pulse inhibition (Hauser et al., 2005) as well as tolerance to the sedative actions of diazepam (van Rijnsoever et al., 2004).
In addition to L-655,708 a number of other imidazobenzodiazepines with binding selectivity for α5- over α1-, α2- and α3-containing GABAA receptors have also been described. These include not only Ro 15-4513 (Hadingham et al., 1993) but also RY 80 (Liu et al., 1996, Skolnick et al., 1997, Opacka-Juffry et al., 1999, Li et al., 2001), RY 023 (Liu et al., 1996, June et al., 2001) and RY 024 (Vergnes et al., 2001, Bailey et al., 2002, McKay et al., 2004). Clearly, such compounds represent valuable tools for further elucidating the functions of the α5 subtype. However, in order to attribute specific behavioural effects of subtype selective compounds to particular GABAA receptor populations it is necessary to be able to discriminate between the levels of occupancy at different GABAA receptor populations in vivo. Thus, although the in vivo binding of radiolabelled Ro 15-1788 has been used to establish the relationship between receptor occupancy and intrinsic efficacy of non-selective benzodiazepines, not only in animals (Facklam et al., 1992) but also in man (Malizia and Richardson, 1995), the non-selective binding affinity of Ro 15-1788 means that it cannot be used as a radioligand to selectively determine the occupancy of a binding-selective compound at a particular GABAA subtype. Therefore, in the present study we evaluated whether [3H]L-655,708 is suitable for specifically labelling α5 subunit-containing GABAA receptors in vivo in a manner analogous to that used to measure occupancy of the combined α1, α2, α3 and α5 subunit-containing GABAA receptor population using [3H]Ro 15-1788 (Atack et al., 1999). Pharmacological and autoradiographic characterisation of the in vivo binding properties of [3H]L-655,708 were used to establish that this ligand bound specifically to GABAA receptors containing an α5 subunit.
Section snippets
Drugs
[3H]L-655,708 was synthesised in-house as described elsewhere (Quirk et al., 1996). This compound is also commercially available from American Radiolabelled Chemicals, Inc. [3H]Ro 15-1788 (70–87 Ci/mmol) was purchased from NEN (PerkinElmer Life Sciences, Boston, MA). Diazepam, flunitrazepam and zolpidem were obtained from RBI (Sigma–Aldrich, Gillingham, UK) and bretazenil was a gift from Roche Labs.
In vitro radioligand binding studies
The affinities of L-655,708, Ro 15-1788, diazepam and zolpidem for the benzodiazepine site of
Affinity of benzodiazepine site ligands
Table 1 shows the affinities of the different benzodiazepine binding site ligands used in the present study. Thus, L-655,708 has 30–70-fold selectivity for α5- compared to α1, α2 and α3-containing GABAA receptors, in agreement with a previous report showing 50–100-fold selectivity (Quirk et al., 1996). In contrast, Ro 15-1788 and diazepam have equivalent affinity at these different GABAA receptor subtypes and are therefore non-selective. On the other hand, zolpidem showed binding selectivity
Discussion
The time course of brain radioactivity clearly demonstrates that [3H]L-655,708 quickly enters the brain and is then rapidly cleared, with maximum brain radioactivity being observed at the earliest time point examined (1 min) and is consistent with previously published data for [3H]L-655,708 (Opacka-Juffry et al., 1999). [3H]Ro 15-1788 has a similar rapid penetration into and clearance from the brain, although in the case of [3H]Ro 15-1788 maximum brain radioactivity occurs after 3 min (Atack et
References (45)
- et al.
Regional differences in the inhibition of mouse in vivo [3H]Ro 15-1788 binding reflect selectivity for α1 versus α2 and α3 subunit-containing GABAA receptors
Neuropsychopharmacology
(1999) - et al.
Effects of hippocampal injections of a novel ligand selective for the α5β2γ2 subunits of the GABA/benzodiazepine receptor on Pavlovian conditioning
Neurobiology of Learning and Memory
(2002) - et al.
Pharmacology of recombinant γ-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated α-subunits
FEBS Letters
(1998) - et al.
Density and pharmacology of α5 subunit-containing GABAA receptors are preserved in hippocampus of Alzheimer's disease patients
Journal of Neuroscience
(2000) - et al.
Drug interactions at GABAA receptors
Progress in Neurobiology
(2002) - et al.
Evaluation of native GABAA receptors containing an α5 subunit
European Journal of Pharmacology
(2001) - et al.
GABAA/benzodiazepine receptor heterogeneity: neurophysiological implications
Neuropharmacology
(1995) - et al.
Which GABAA-receptor subtypes really occur in the brain?
Trends in Neurosciences
(1996) - et al.
Evaluation of [methyl-3H]L-655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABAA receptors containing α5 subunit
Nuclear Medicine and Biology
(1999) - et al.
GABAA receptors: immunocytochemical distribution of 13 subunits in the adult brain
Neuroscience
(2000)
[3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the α5 subunit
Neuropharmacology
Behavioural pharmacology of zolpidem relative to benzodiazepines: a review
Pharmacology, Biochemistry, and Behavior
Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain
Brain Research
Differential sensitivity to inverse agonists of GABAA/benzodiazepine receptors in rats with genetic absence-epilepsy
Epilepsy Research
A single histidine in GABAA receptors is essential for benzodiazepine agonist binding
Journal of Biological Chemistry
International Union of Pharmacology. XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function
Pharmacological Reviews
In vivo interaction of zolpidem with central benzodiazepine (BZD) binding sites (as labeled by [3H]Ro 15-1788) in the mouse brain. Preferential affinity of zolpidem for the ω1 (BZD1) subtype
Journal of Pharmacology and Experimental Therapeutics
Comparative in vivo and in vitro regional selectivity of central ω (benzodiazepine) site ligands in inhibiting [3H]flumazenil binding in the rat central nervous system
Journal of Pharmacology and Experimental Therapeutics
θ, A novel γ-aminobutyric acid type A receptor subunit
Proceedings of the National Academy of Sciences of the United States of America
6,7-Dihydro-2-benzothiophen-4(5H)-ones: a novel class of GABA-A α5 receptor inverse agonists
Journal of Medicinal Chemistry
Identification of a novel, selective GABAA α5 receptor inverse agonist which enhances cognition
Journal of Medicinal Chemistry
Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction
Biochemical Pharmacology
Cited by (26)
Combined treatment with exercise and α5GABA<inf>A</inf>R inhibitor promotes motor function recovery after intracerebral hemorrhage
2022, Neuroscience LettersCitation Excerpt :To specifically inhibit α5GABAAR, we intraperitoneally administered L-655,708 (product#:1327, R&D Systems, Inc., USA) to the ICH + L6 and ICH + L6EX groups at a dose of 0.5 mg/kg. L-655,708 has a 30- to 70-fold higher affinity for α5 subunit-containing GABAA receptors than for other GABAA receptor subtypes, and this dose (0.5 mg/kg) is known to be high enough to inhibit α5GABAAR, yet it is low enough to minimize inhibition of other GABAA receptor subtypes [24]. L-655,708 was first dissolved in dimethyl sulfoxide and then diluted to the final volume with physiological saline.
Comparison of rapid and long-lasting antidepressant effects of negative modulators of α5-containing GABA<inf>A</inf> receptors and (R)‑ketamine in a chronic social defeat stress model
2018, Pharmacology Biochemistry and BehaviorCitation Excerpt :The GABAA (γ‑aminobutyric acid, type A) receptors play a role in a number of psychiatric disorders including depression since the regulation of GABAA receptors is known to influence glutamate neurotransmission (Kalueff and Nutt, 2007; Luscher et al., 2011; Rudolph and Knoflach, 2011; Rudolph and Möhler, 2014). Recent studies showed that two negative allosteric modulators (NAMs: L-655,708 and MRK-016) (Atack et al., 2005, 2006, 2009) of α5 subunit-containing GABAA receptors produced rapid antidepressant effects in chronic restraint stress (CRS) and chronic unpredictable stress (CUS) models (Fischell et al., 2015). Unlike (R,S)‑ketamine, MRK-016 produced no impairment of rota-rod performance, no reduction of prepulse inhibition, no conditioned-place preference, and no change in locomotion (Zanos et al., 2017).
Emerging treatment mechanisms for depression: Focus on glutamate and synaptic plasticity
2016, Drug Discovery TodayCitation Excerpt :The authors tested a GABA-A alpha-5 selective inverse agonist, L-655,708 (FG-8094), which has approximately 30-fold selectivity for alpha-5 over other GABA-A receptors containing alpha-1, alpha-2, or alpha-3. The results indicate that the compound is a partial allosteric modulator at GABA-A receptors containing all the alpha subunits previously mentioned, with comparable efficacy albeit with higher affinity for alpha-5 [95,96]. Therefore to further examine the efficacy of targeting alpha-5 subunit, they tested another apha-5 agent, MRK-16.
Evaluation of a novel series of fluorine-18-labeled imidazobenzodiazepines as potential new positron emission tomography radioligands for the GABA<inf>A</inf> receptor
2014, Nuclear Medicine and BiologyCitation Excerpt :Bretazenil (Sigma-Aldrich, #B6434) is an anxiolytic drug derived from the benzodiazepine family, and is known to block all benzodiazepine binding sites. Bretazenil was chosen for these studies as this compound has been extensively used in similar published studies [15–18]. The general protocol was the same for all the ligands studied.
Measurement of the pharmacokinetics and pharmacodynamics of neuroactive compounds
2010, Neurobiology of Disease