Perspectives in Pharmacology A New Benzodiazepine Pharmacology

Classical benzodiazepine drugs are in wide clinical use as anxiolytics, hypnotics, anticonvulsants, and muscle relaxants. They act by enhancing the -aminobutyric acidA (GABAA) receptor function in the central nervous system. The pharmacological relevance of the multitude of structurally diverse GABAA receptor subtypes has only recently been identified. Based on an in vivo point mutation strategy, 1-GABAA receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection, whereas 2-GABAA receptors, but not 3-receptors, mediate anxiolysis. Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the new subtype-selective drugs. Promising new anxiolytics have already been developed. A new pharmacology of the benzodiazepine site is on the horizon. GABAergic inhibition is one of the most rapidly developing topics in neuropharmacology. New therapeutic opportunities arise due to increasing insights into the molecular architecture and diversity of the components involved in signal transduction such as GABAA receptors, GABAB receptors, and GABA transporters (Fig. 1). GABAA receptors are important drug targets representing the sites of action of benzodiazepines, barbiturates, and neurosteroids. The present article focuses on the pharmacological distinction of GABAA receptor subtypes as a basis for the development of new drugs that target restricted neuronal networks. In particular, new ligands of the benzodiazepine site acting selectively on GABAA receptor subtypes are expected to dissect the pharmacological spectrum of classical benzodiazepines and display a minimum of side effects. For further information on GABAA receptor subtypes other recent reviews may be consulted (Barnard et al., 1998; Möhler et al., 2000; Olsen and Homanics, 2000; Whiting et al., 2000; Möhler, 2001). Synaptic Action of Benzodiazepines At synapses, GABAA receptors are activated by a brief nonequilibrium exposure to high concentrations of GABA. Consistent with an increase in the affinity of the receptors for GABA, therapeutically active benzodiazepines prolonged the decay of spontaneous miniature inhibitory postsynaptic currents (mIPSC). Similarly, the amplitude of mIPSC was found to be enhanced by a benzodiazepine agonist in various neuronal systems (Perrais and Ropert, 1999; Hajos et al., 2000), suggesting that the drug-induced increase of the affinity for GABA resulted in the recruitment of more receptors for activation by GABA. However, in other neuronal systems the amplitude of the mIPSC remained unaltered by a benzodiazepine agonist (Mody et al., 1994; Poncer et al., 1996; Hajos et al., 2000), which has been interpreted to indicate that the release of a single quantum of GABA saturates all of the available GABAA receptors in the respective synapses inducing a maximal peak response without further enhancement by the drug. Thus, at synapses that generate mIPSCs, the postsynaptic receptor occupancy by GABA appears to be celland synapse-specific, reflecting local differences in the number of receptors or the GABA concentration in the cleft. Accordingly, the influence of benzodiazepine agonists on the amplitude of mIPSCs appears to vary with the operational configuration of the GABAergic synapse (Hajos et al., 2000). In summary, the enhancement of a GABAergic inhibitory response by a benzodiazepine agonist is based on the prolonged decay of the mIPSC and a potential increase of the mIPSC amplitude. Even if the peak mIPSC amplitude is not enhanced per se, the drug-induced prolongation of individual mIPSCs will be reflected in an increased peak amplitude of the compound inhibitory response caused by the summation of several miniature currents (Mody et al., 1994). ABBREVIATIONS: GABA, -aminobutyric acid; mIPSC, miniature inhibitory postsynaptic currents. 0022-3565/02/3001-2–8$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 900028/952622 JPET 300:2–8, 2002 Printed in U.S.A. 2 at A PE T Jornals on Sptem er 4, 2017 jpet.asjournals.org D ow nladed from GABAA Receptors and Their Multiplicity Based on the presence of 7 subunit families comprising at least 18 subunits in the central nervous system ( 1–6, 1–3, 1–3, , , , 1–3,) the GABAA receptors display an extraordinary structural heterogeneity. Most GABAA receptor subtypes in vivo are believed to be composed of -, -, and subunits. The role of the -, -, and subunits, which have a very limited expression pattern in the brain, remains to be determined, but it is possible that they substitute for the -subunit in combinations. The physiological significance of the structural diversity of GABAA receptors lies in the provision of receptors that differ in their channel kinetics, affinity for GABA, rate of desensitization, and subcellular positioning. In addition, the GABAA receptor subtypes can be distinguished by their pharmacology. Synaptic and Extrasynaptic GABAA Receptors. The first electron microscopic studies of GABAA receptors revealed the ubiquitous presence of extrasynaptic GABAA receptors in the cerebellum, thalamus, and cerebral cortex (Somogyi, 1989). In fact, synaptic GABAA 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 GABAA receptor subtypes to different types of synapses. For instance, the 2 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 6 subunit, which represents diazepam-insensitive GABAA 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, GABAA receptors containing the -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 GABAA receptors differ in their kinetic properties in line with their distinct functional roles. For instance, extrasynaptic GABAA receptors containing the -subunit in dentate gyrus and cerebellum are tailormade 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. GABAA receptors containing the 2-subunit are postsynaptic and characterized by rapid desensitization kinetics, whereas extrasynaptic GABAA receptor containing the 3-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 GABAA 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 GABAA 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 GABAA receptors. Gephyrin was shown to be required, along with the 2-subunit, for postsynaptic clustering of major GABAA 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 GABAA 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 GABAA Receptors. Receptors containing the 1-, 2-, 3-, or 5subunits in combination with any of the -subunits and the 2-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 1 2 2, 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). Receptors containing the 2or 3subunit are considerably less abundant and are highly expressed in brain areas where the 1-subunit is absent or present at low levels (Table 1). The 2and 3-subunits are frequently coexpressed with the 3and 2-subunits, which is particularly evident in hippocampal pyramidal neurons ( 2 3 2) and in cholinergic neurons of the basal forebrain ( 3 3 2). The ligand-binding profile of these receptors differs from that of 1 2 2 by having a considerably lower displacing potency for ligands such as 3-carboxymethoxy-carboline, CL 218,872, and zolpidem (Table 1). Receptors containing the 5-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 GABAA receptor population, predominantly coassembled with the 3-and 2-subunits. The 5-receptors are differentiated from 1 2 2, 2 3 2, and 3 3 2 receptors by a lower affinity to CL 218,872 and near insensitivity to zolpidem (Table 1). Fig. 1. Scheme of a GABAergic synapse depicting the major elements of signal transduction. GABAA receptors are indirectly linked to the synaptic anchoring protein gephyrin. Benzodiazepine Pharmacology 3 at A PE T Jornals on Sptem er 4, 2017 jpet.asjournals.org D ow nladed from