Synaptic and Extrasynaptic GABA_A Receptors.

The first electron microscopic studies of GABA_Areceptors revealed the ubiquitous presence of extrasynaptic GABA_A receptors in the cerebellum, thalamus, and cerebral cortex (Somogyi, 1989). In fact, synaptic GABA_A receptors are best seen using postembedding electron microscopy, or by immunofluorescence staining using weakly fixed brain sections (Fritschy et al., 1998a), showing pronounced enrichment in the postsynaptic density of GABAergic synapses compared with extrasynaptic sites (Nusser et al., 1995). These studies also revealed differential targeting of GABA_A receptor subtypes to different types of synapses. For instance, the α₂ subunit in hippocampal pyramidal cells is concentrated in synapses on the axon-initial segment (Nusser et al., 1996a; Fritschy et al., 1998a), as well as in synapses formed by cholecystokinin-positive basket cells on the soma (Nyı́ri et al., 2001). The α₆ subunit, which represents diazepam-insensitive GABA_A receptors in the cerebellum can be found both in excitatory and inhibitory synapses in glomeruli of the granule cell layer, as well as extrasynaptically (Nusser et al., 1996b,1999; Sassoè-Pognetto et al., 2000). Finally, GABA_A receptors containing the δ-subunit in the cerebellum are exclusively found at extrasynaptic sites (Nusser et al., 1998). Both extrasynaptic receptor types mediate tonic inhibition of neuronal activity (Brickley et al., 1996, 2001).

Synaptic and extrasynaptic GABA_A receptors differ in their kinetic properties in line with their distinct functional roles. For instance, extrasynaptic GABA_Areceptors containing the δ-subunit in dentate gyrus and cerebellum are tailor-made for tonic inhibition, due to their high affinity for GABA and slow desensitization kinetics (Brickley et al., 1996; Mody and Nusser, 2000). Marked differences in desensitization kinetics have also been reported for synaptic and extrasynaptic receptors in inferior olivary neurons. GABA_A receptors containing the α₂-subunit are postsynaptic and characterized by rapid desensitization kinetics, whereas extrasynaptic GABA_A receptor containing the α₃-subunit desensitize very slowly (Devor et al., 2001). Interestingly, such differences are not evident in recombinant expression systems, suggesting the contribution of additional factors regulating GABA_A receptor function at synaptic and extrasynaptic sites. A further identification of the molecular composition of GABAergic postsynaptic densities is expected to shed light on the functional regulation of synaptic GABA_A receptors (Moss and Smart, 2001).

The demonstration that gephyrin, a synaptic clustering protein initially isolated with glycine receptors, is present in a subset of GABAergic synapses in the retina (SassoèPognetto et al., 1995) prompted the analysis of its role in relation to GABA_A receptors. Gephyrin was shown to be required, along with the γ₂-subunit, for postsynaptic clustering of major GABA_A receptor subtypes (Essrich et al., 1998; Kneussel et al., 1999). A recent study demonstrated by immunoelectron microscopy and double-immunofluorescence staining that gephyrin is colocalized with the vast majority of postsynaptic GABA_A receptors throughout the central nervous system (Sassoè-Pognetto et al., 2000), indicating that it represents a useful marker of GABAergic (and glycinergic) synapses.

Diazepam-Sensitive GABA_A Receptors.

Receptors containing the α₁-, α₂-, α₃-, or α₅- subunits in combination with any of the β-subunits and the γ₂-subunit are most prevalent in the brain (Fig. 2). These receptors are sensitive to benzodiazepine modulation. The major receptor subtype is assembled from the subunits α₁β₂γ₂, with only a few brain regions lacking this receptor (granule cell layer of the olfactory bulb, reticular nucleus of the thalamus, spinal cord motoneurons) (Fritschy and Möhler, 1995; Pirker et al., 2000) (Table 1).

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Figure 2

The four classes of diazepam-sensitive GABA_A receptors are distinguished by the type of α-subunit (α₁, α₂, α₃, α₅) and their largely distinct neuronal localizations as demonstrated immunohistochemically in mouse brain sections. The major known pharmacological actions mediated via the respective receptor subtypes are indicated.

Receptors containing the α₂- or α₃- subunit are considerably less abundant and are highly expressed in brain areas where the α₁-subunit is absent or present at low levels (Table 1). The α₂- and α₃-subunits are frequently coexpressed with the β₃- and γ₂-subunits, which is particularly evident in hippocampal pyramidal neurons (α₂β₃γ₂) and in cholinergic neurons of the basal forebrain (α₃β₃γ₂). The ligand-binding profile of these receptors differs from that of α₁β₂γ₂ by having a considerably lower displacing potency for ligands such as 3-carboxymethoxy-β-carboline, CL 218,872, and zolpidem (Table1).

Receptors containing the α₅-subunit are of minor abundance in the whole brain (Table 1) but are expressed to a significant extent in the hippocampus, where they comprise 15 to 20% of the diazepam-sensitive GABA_A receptor population, predominantly coassembled with the β₃-and γ₂-subunits. The α₅-receptors are differentiated from α₁β₂γ₂, α₂β₃γ₂, and α₃β₃γ₂receptors by a lower affinity to CL 218,872 and near insensitivity to zolpidem (Table 1).

The γ₁- and γ₃-subunits characterize a small population of receptors that contain various types of α- and β-subunits. Due to their reduced affinity for the classical benzodiazepines, they do not appear to contribute to any great extent to their pharmacology in vivo (Möhler et al., 2000;Whiting et al., 2000; Möhler, 2001).

Diazepam-Insensitive GABA_A Receptors.

GABA_A receptors that do not respond to clinically used ligands, such as diazepam, flunitrazepam, clonazepam, and zolpidem, are of low abundance in the brain and are largely characterized by the α₄- and α₆-subunits (Table 1). Receptors containing the α₄-subunit are generally expressed at very low abundance but more prominently in thalamus and dentate gyrus (Pirker et al., 2000); those containing the α₆-subunit are restricted to the granule cell layer of the cerebellum (about 30% of all GABA_A receptors in the cerebellum). Both receptor populations are structurally heterogeneous, and the majority of the α₆-containing receptors are of the α₆β₂γ₂combination (Table 1). The benzodiazepine site profile of α₄- and α₆-receptors is characterized by a low affinity for flumazenil and bretazenil and a switch in the efficacy of Ro 15-4513 (ethyl 8-acido-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-α] [1,4]benzodiazepine-3-carboxylate) from an inverse agonist to an agonist. The δ-subunit is frequently coassembled with the α₄- or the α₆-subunit in benzodiazepine insensitive receptors (Möhler et al., 2000;Whiting et al., 2000; Möhler, 2001).

In the retina, homomeric receptors consisting of the ρ-subunit represent a particular class of GABA-gated chloride channels. Their GABA site is insensitive to bicuculline and baclofen, and they are not modulated by barbiturates or benzodiazepines (Table 1). Due to these distinctive features the receptors are frequently termed GABA_c receptors (Bormann, 2000), although they can also be considered as a homomeric class of GABA_A receptors (Barnard et al., 1998).

Receptors Mediating Sedation.

Sedation is a major property of many benzodiazepine site ligands and has now been shown to be mediated via α₁-receptors. Among α₁-, α₂-, and α₃-point-mutated mice only the α₁(H101R) mutants were resistant to the depression of motor activity by diazepam and zolpidem (Rudolph et al., 1999; Crestani et al., 2000a; Löw et al., 2000). This effect was specific for ligands of the benzodiazepine site since pentobarbital or a neurosteroid remained as effective in α₁(H101R) mice as in wild-type mice in reducing motor activity. An α₁(H101R) mouse line was also generated by McKernan et al.(2000). When measured under novelty (“stress”) conditions, diazepam increased locomotion compared with wild-type (McKernan et al., 2000). This effect is apparently dependent on the test procedure and can be induced also in the α₁(H101R) mice generated by Rudolph et al. (1999) under comparable test conditions (Crestani et al., 2000b).

Receptors Mediating Amnesia.

Anterograde amnesia is a classical side effect of benzodiazepine drugs. The memory-impairing effect of diazepam, analyzed in a step-through passive avoidance paradigm, was strongly reduced in the α₁(H101R) mice compared with wild-type mice as shown by the increased latency for reentering the dark compartment 24 h after training (Rudolph et al., 1999). This effect was not due to a potential nonspecific impairment since the ability of a muscarinic antagonist to induce amnesia was retained in the α₁(H101R) mice. These results demonstrate that the diazepam-induced anterograde amnesia is mediated by α₁-receptors.

Receptors Providing Protection Against Seizures.

The anticonvulsant activity of diazepam, assessed by its protection against pentylenetetrazole-induced tonic convulsions, was reduced in α₁(H101R) mice compared with wild-type mice (Rudolph et al., 1999). The anticonvulsant effect of diazepam, which remained in the α₁(H101R) mice, was due to GABA_A receptors other than α₁, since it was antagonized by flumazenil. Sodium phenobarbital remained fully effective as an anticonvulsant in α₁(H101R) mice with a dose response intensity similar to that of wild-type mice. These results show that the anticonvulsant activity of benzodiazepines is partially but not fully mediated by α₁-receptors. The anticonvulsant action of zolpidem is exclusively mediated by α₁-receptors, since its anticonvulsant action is completely absent in α₁(H101R) mice (Crestani et al., 2000a).

Receptors for Anxiolysis.

New strategies for the development of daytime anxiolytics that are devoid of drowsiness are of high priority. The particular receptor subtype that mediates the anxiolytic activity of benzodiazepine drugs was recently identified (Löw et al., 2000). When the reactivity to naturally aversive stimuli is measured in wild-type mice, diazepam increases the time spent in the lit area of the light/dark choice test and on the open arms of the elevated plus maze, respectively. In contrast, the α₂-point-mutated mice were resistant to the effect of diazepam in these test paradigms (Löw et al., 2000). This lack of response was specific for ligands of the benzodiazepine site since α₂(H101R) mice retained the ability to display an anxiolytic-like response to sodium phenobarbital. Thus, the anxiolytic-like action of diazepam is selectively mediated by the enhancement of GABAergic transmission in a population of neurons expressing the α₂-GABA_Areceptors. Thus, the α₂-GABA_A receptors are highly specific targets for the development of future selective anxiolytic drugs. They represent only about 15% of all diazepam-sensitive GABA_A receptors. Selective ligands are therefore expected to be devoid of the major side effects that afflict the classical benzodiazepine anxiolytics.

It had previously been assumed that the anxiolytic action of diazepam is based on the dampening of the reticular activating system. It is mainly represented by noradrenergic and serotonergic neurons of the brain stem, which express exclusively α₃-receptors. The analysis of the α₃-point-mutated mice [α₃(H126R)] indicated that the anxiolytic effect of benzodiazepine drugs, measured as described above, is not mediated by α₃-receptors (Löw et al., 2000). The reticular activating system therefore does not appear to be a major contributor to anxiolysis. In contrast, the α₂-GABA_A receptors are highly expressed in cells in the cerebral cortex and the hippocampus, including their pyramidal cells, which display particularly high densities of α₂-GABA_Areceptors on their axon initial segment (Nusser et al., 1996a; Fritschy et al., 1998a). Thus, controlling the output of these principal neurons may contribute to anxiolysis.

Receptors Mediating Myorelaxation.

The degree of muscle tone can be assessed in the horizontal wire test, in which the ability of the animals to grasp and hang onto a wire is measured. The muscle relaxant effect of diazepam is largely mediated by α₂-GABA_A receptors, as shown by the failure of diazepam to induce changes in muscle tone in the α₂-mutated mouse line (Crestani et al., 2001). It was only at high doses that α₃-receptors were also implicated. Besides the limbic system (see above), α₂-receptors are highly specifically expressed in the spinal cord, notably in the superficial layer of the dorsal horn and in motor neurons (Bohlhalter et al., 1996) with the latter being most clearly implicated in muscle relaxation. It is important to note that the muscle relaxant effect requires higher doses of diazepam than its anxiolytic activity. This is attributed to a higher receptor occupancy required for muscle relaxation.

L-838,417.

The benzodiazepine site ligand L-838,417 interacts with comparable affinity with α₁-, α₂-, and α₃-receptors and with only 3-fold lower affinity with α₅-receptors. However, it displays a dramatic subtype-selective efficacy. L-838,417 fails to modulate the GABA response at α₁-receptors but enhances the GABA response at α₂-, α₃-, and α₅-receptors (McKernan et al., 2000). In line with a lack of α₁-receptor activation, L-838,417 showed a high potency in anxiolytic tests (elevated plus maze and fear-potentiated startle) and in anticonvulsant tests (pentylenetetrazole, audiogenic seizures). However, even at doses that occupied 95% of benzodiazepine sites, L-838,417 failed to impair motor performance (rotarod test, chain-pulling test) (McKernan et al., 2000). These findings are a major step forward. Ligands with subtype-selective efficacy, which distinguish α₂-, α₃-, and α₅,-receptors from α₁-receptors, provide a new way to develop selective anxiolytics without a sedative component. Further improvement may be achieved by focusing the ligand affinity or efficacy more specifically on α₂-receptors (see above).

SL65.1498.

The pyrido-indole-4-carboxamide derivative SL65.1498 (6-fluoro-9-methyl-2phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) shows higher affinity for α₁-, α₂-, and α₃-GABA_A receptors compared with α₅-receptors. In addition, it acts as full agonist at α₂- and α₃-receptors but as a partial agonist at α₁-GABA_A receptors. In line with its selectivity for the activation of α₂- and α₃-receptors, the compound showed potent anxiolytic action in animal models (punished lever pressing, punished drinking, elevated plus maze, light/dark test) but did not impair motor coordination (e.g., rotarod) or working memory (Morris water maze) (Scatton et al., 2000).

Zaleplon.

Zaleplon (CL 284,846) is a pyrazolopyrimidine developed for the treatment of insomnia (Sanger et al., 1996). At recombinant receptors, zaleplon binds preferentially to α₁-receptors (α₁β₂γ₂) and to receptors containing the γ₃-subunit but binds 8- to 20-fold less to α₂-, α₃-, and α₅-receptors (Dämgen and Lüddens, 1999). Thus, zaleplon is largely a ligand with preference for α₁-receptors, which is in keeping with its preponderant hypnotic activity. The contribution of its interaction with γ₃-receptors is unclear since these receptors are of low abundance in the brain.