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ultiple benzodiazepine eceptors: no reason for anxiety Adam Doble and Ian L. Martin Since the introduction of the benzodiazepines

info clinical pracfice in 1960, these drugs have been widely employed as anxiolytics, sedative/hypnotics and anticonvulsants. In recent years, concern has been expressed about their sideeffecfs, and their use has declined. During this latter period many advances have been made in understanding the molecular mechanisms by which these

drugs produce their effects. Adam Doble and Ian Martin review this progress and highlight the possibilities that these advances may hold for the development of more efficacious and specific medicines. The fifteen years that have followed the discovery of receptors for benzodiazepines in the mammalian CNS have witnessed great progress in our understanding of the molecular mechanisms underpinning the interaction of these txanquillizers with GABA-mediated transmission, as well as in the development of novel nonbenzodiazepine drugs acting via the GABA system. Paradoxically, these fundamental advances have been accompanied by a progressive fall from grace of the benzodiazepines as therapeutic agents in the treatment of anxiety, due to the increasing unacceptability of their side-effects (sedation, dizziness, interactions with alcohol and, particularly, the risk of dependence with long-term use). Not unnaturally, these two processes have reinforced each other, and given rise to the hope that some of the novel non-benzodiazepine tranquillizers might be devoid of the unwanted sideeffects associated with the classical benzodiazepines. One of the more attractive hypotheses to have arisen in this context is that of benzodiazepine receptor multiplicity. According to this idea, different receptor subtypes would exist in different A. DobJe is Director of the Neurochemistry

Departmentat Rhdne-PoulencRarer, Centre

de Recherche de Vitry-AJforfviJJe, 13 Quni 1uJes Guesde- BP 14,94403 Vity sur Seine, Cedex, France. 1. L. Martin is a Senior Scientist in the MolecularNeurobiologyUnit, MRC Centre, HiJJs Road, Cambridge CB2 2QH, UK. His address from Zst February 1992 wiJJbe Departmentof Pharmacology, Faculty of Medicine. University of Alberta, Edmonton. Alberta, Canada T6G 2H7. @ 1992.Ekwier

Science Publkhen

Ltd (UK)

brain areas, subserving different physiological functions. It should then be possible to develop molecules selective for these different subtypes that would show only part of the spectrum of behavioural effects seen with the benzodiazepines. When this hypothesis was first proposed in the early 198Os, the evidence in favour of multiple benzodiazepine receptors was essentially indirect’,‘. However, more recent advances in both mol-

ecular biology (cloning, in situ hybridization, re-expression and mutation of benzodiazepine receptors) and medicinal chemistry (synthesis of a huge variety of novel agents interacting with the benzodiazepine receptor) enable us directly to address the question as to whether it will be possible to develop behaviourally selective drugs by creating molecules with a predetermined receptor subtype selectivity. Origin of BZ1 and BZ2 Two types of benzodiazepine receptor were originally proposed on the basis of pharmacology and distribution. The BZ1 subtype, found throughout the brain but predominant in the cerebellum, and the BZz subtype found principally in the cortex, hippocampus and spinal cord, were suggested to subserve different physiological functions. Three lines of evidence, albeit indirect, were compatible with this hypothesis: firstly, the triazolopyridazine CL218872, and the P-carboline P-CCE, displaced [3H]benzodiazepine binding in the hippocampus with shallow slopes on the Hill plot, suggesting

Fig. 1. Above: Distribution of BZ, and BZ, receptors determined by autoradiography. Binding of [3H]suriclone (7.5 nM) to sagina/ sections of rat brain. (a) To&J binding. (b) Binding in the presence of aJpidem (300 nM) shows BZ, receptors. (c) Subtracting (b) from (a) gives the distribution of BZ, receptors. (d) Nonspecific binding in presence of fiumazenil (1 j@. Kindly provided by Dr C. Malgouris. Right: Patterns of a-subunit gene expression in rat brain as determined by in situ hybridization. Parallel sagittal rat brain sections hybridized wifh 35S-iabelled antisense oligonucleotides specific for d-d genes of Ihe GAB4 receptor. A, amygdaloid area; BS, brain stem; CPU, caudate pufamen; Cl, c/a&rum; Cb, cerebellum; DLG, dorsal JaJeraJgeniculate thalamic nucleus; GP, globus pallidus; Lat, lateral deep oerebellar nucleus; MQN, medial genicuJaJe thalamic nucleus; ZI, zona incerta; Rt, reticular thalamic nucleus; VP, irentral pallidurn; S, subiculum. Scale bar = 2 mm. Data kindly provided

by authors of Ref. 23.

TiPS - Februay

1992 [Vol. 231

the existence of a mixed population of sites3f4. B-Carbolines have also been shown to interact with preferentially benzodiazepine receptors in the cerebellum in vivo5. Secondly, Lippa et a1.6 suggested that CL218872 had anxiolytic effects but was only weakly sedative, and proposed that this was due to preferential interaction with the BZr site. Finally, photoaffinity labelling studies with [3H]flunitrazepam showed that at least four proteins, with molecular masses of 51,53,55 and 59 kDa, were photolabelled with this compound’. The regional distribution of these proteins in the rat brain corresponded to that predicted for BZr and BZz sites, with the lower molecular mass protein showing the highest affinity for CL218872 (Ref. 7). However, these latter studies suggested that the heterogeneity of the receptor population was greater than first thought. a-Subunit heterogeneity A possible molecular for this benzodiazepine heterogeneity has been by the cloning of cDNAs multiple isoforms of a,

substrate receptor provided encoding B and y

77

subunits that are thought to constitute the GABA* receptors (see Box). Three pieces of evidence suggest that it is the particular (Y subunit isoform present in the receptor oligomer that defines recognition characteristics of the benzodiazepine site. The proteins photolabelled with [3H]flunitrazepam exhibited a deferential topographical localization in the CNS. The cerebellum, an area rich in BZr receptors, contained only one labelled protein with a molecular mass of 51 kDa, while in other brain regions that contain significant amounts of BZ2 type receptors, such as the cortex and hippocampus, additional photolabelled proteins of higher molecular mass were found’. Comparative immunoblotting studies demonstrated that all the proteins covalently labelled with [3H]~unitrazepam were cr subunits; the B subunits were not so modifiedsl*. Subsequent studies with subunitspecific antibodies have shown that the 51 kDa protein corresponds to al, the 53 kDa protein to a2 and the 59 kDa protein to a3 (Refs 12, 13). The pharmacological l

characteristics of these photolabelled proteins, and their topographical brain distribution, are thus compatible with the differential distribution of +ho . .._ hanroY_ diazepine receptor subtypes. l Using a completely different Pritchett and co%!z’ also concluded that a-subunit heterogeneity underlies benzodiazepine receptor multiplicity. analysed the They pharmacological specificity of recombinant GABA* receptors containing a single a-subunit isoform. Different human a isoforms were coexpressed with Bl and y2 in cells of the human kidney 293 cell line, and the binding characteristics of the GABA* receptors found in the cell membrane were evaluated. Receptors containing the al subunit displayed the BZr phenotype (high affinity for CL218872 and the B-carbolines), whilst those containing a2 or (u3 displayed a BZz phenotype. l Site-directed mutagenesis experiments have pinpointed the region within the a subunit that is critical in determining the BZr/ BZz phenotype. Substitution of a single glutamic acid residue in the rat a3 sequence by glycine (which is found at the corresponding position 201 of the al sequence) confers on the hybrid receptor high affinity for CL218872 and zolpidem15. Multiple a subunits: beyond BZ,/BZ, Experiments both with recombinant receptors and with photolabelled receptors indicated that, although the BZr subtype seemed to correspond to receptors containing al, the BZz subtype represented a heterogeneous population of sites containing a2 or a3 subunits (see Fig. 1). The situation became more complicated with the discovery of the a5 subunit; recombinant receptors containing this isoform displayed the BZ2 phenotype, but differed from those containing either 012 or ar3. in having an extremely low affinity for the imidazopyridine zolpidem16. GABA* receptors containing the a6 isoform, such as are found only in the granule cell layer of the cerebellum17, exhibit very low affinities for the classical benzodiazepines. The only compound to be recognized with high af-

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78

Subunit structure of native GABA* receptors in the mammalian CNS inspection of the deduced ammo acid sequencer from the subunits obtained from mot&ar

doning

SludieS

the GAFtA* receptor suggest that they each contain an N-texminal segment of about 200 amino acids before the first of four hydrophobic, putative membranespanning, segments (Ml-M4). Each of these hydrophobic domains is connected to its neighbours by short hydrophilic segments of amino acids, with the exception oftbe segment between M3 and M4, which is larger &tl~

and can contain consensus phosphorylationsitesr. This organization is very similar to that shown by subunits of the nicotinic acetykholine receptor, the archetypal member of this family of ligand-gated ion channel receptors, for which a significant amount of structural information is available.

The nicotinic acetylcholine receptor from both Tor-

pedo and the mammaRan CNS is composed of five

subunits; in the former case the stoichiometry and order are known to be or, 8, or,y, 6 (see Ref. 2). It would thus seem probable that the GABAA receptor also exhibits pseudo-pentameric symmetry. However, in the case of both the GABAA and nicotinic acetylcholine receptors of the mammalian CNS there appear to be multiple genes encoding d&rent isoforms of the individual subunits. At the present time the ammo acid sequences of six or, four g, three y, one 6 and a p subunit(s) have been deduced fmm molecular cloning studies. Further heterogeneity is apparent within certain subunit isoforms due to alternative splicing (see Ref. 3). and more

isoforms could yet be found, recombinants of which may compromise the interpretations that have been rehearsed here.

It is the ar subunits of the nicotinic acetylcholine receptor that are responsible for the recognition of aoztykholine. Photoaffimty labelling studies with muscimol suggest that the recognition site for GABA is present on the l3subunit of the GABAA receptor4.5. Both the GABA and nicotinic acetylcholine receptors exhibit positive cooperativity in response to agonists, which suggests that two ft subunits must be present in the native GABA* receptor complexes. However, some recombinant studies indicate that GABA responses can be obtained in the absence of fl subunit expression,

its inclusion does appear to have an effect on the desensitizationcharacteristics of the responseh7.

dtkmgh

It is clear that in order to obtain responses to benzodiazepinerecognition-site ligands, a y subunit is es~ential8.Recombinantscontaining e, piand y2 (where i=1-6, j=14) appear to exhibit an appropriate pharma-

cology with agonist, antagonist and inverse agonist @rids. If the YZsubunit in these studies is replaced by

firdty by receptors containing ar6 was the partial inverse agonist sarmazenil (Ro154513), which can thus be used as a selective marker for the BZ& phenotype of the GABAA receptor1sJ9. Functionally, the BZ& isoform may be involved in the pharrnacological effects of ethanol. Sarmazenil has been claimed to reverse certain narcotic effects of ethanol” I and the recognition

the y1 subunit, then not only is the assembly of the functional complex less efficient, as judged by [3H]flunitrazepam binding capacity measurements, but the affinities for antagonists, inverse agonists and antagonists are markedly reducedg. The functional importance of the 6 and p subunits is uncertain. The 6 subunit, identified by cDNA cloning techniqueslO, appears to exhibit a particularly high density in the rat cerebellum according to immunohi&chemical studies”. The p subunit, originally cloned from a human retina library, exhibits GABAactivated currents when expressed alone in Xenopus oocytes; the effects of the benzodiazepines were not investigated=. Functional studies with the barbiturates and the steroids have not been widely documented in recombinant studies. These compounds exhibit robust responses with native GABAA receptors by interacting with distinct recognition sites on the complex13. There is a rapidly increasing body of evidence concerning the functional consequences of inclusion, or exclusion, of individual subunit isoforms within recombinant receptors. In no case has the protein expressed been determined. The pharmacological signature of particular subunit recombinations has not been addressed within the mammalian CNS. These comparisons are necessary but wilI require considerable efforts, and they must be interpreted with care; the species used and the stage of its ontogenic development will be important.

References 1 Barnard, E. A., Darlison, M. G. and Seeburg, P. H. (1967)

TrendsNeurosci.10,502-509 2 Unwin, N. (1989) Neuron3,66%176 3 Liiddens,H. and Wisden, W. (1991) TrendsPhurmucol.Sci. 12,49-51 4 Casalottt,S. O., Stephenson,F. A. and Barnard, E. A. (1986) 1. Biof. Chem. 261,150X3-15016 5 Dem. L.. Ranson. R. W. and Olsen, R. W. (1986) Biochenr. ’ . Biop&s. Rec. Co&nun. 138,13&X-1314 6 Verdoom, T. A., Drawbn, A., Ymer, S., Seebum, P. H. and

-Sakmann, B. (1990) neuron 4,919-928 7 Sigel, E., Baur, R., Trube, G., Miihler,H. and Malberbe, P. (1990) Neuron 5,703-711 8 Pritchett, D. B. et al. (1989) Nature 338, 582-585 9 Ymer, S. et al. (1990) EMBO j. 9,3261-3267 10 Shivers, 8. D. et al. (1989) Neuron 3,327-337 11 Henke, D., Mertens, S.,. Trzeciate, A., Gillessen, D. and M&ler, H. (1990) FEBS Lett. 283,145-149 12 Cutting, G. R. et al. (1991) Proc. Natl Ad. Sci. USA 88, 2673-2677 13 Peters, J. A., Kirkness, E. F., Callacban, H., Lambert, J. J. and Turner, A. J. (1988) Br. 1. Pharmacol. 94, X257-1269

properties of [3H]sarmazenilbinding sites in the cerebellum are modified in a strain of rats outbred for their sensitivity towards alcoholzl. The neurochemical and electrophysiological studies carried out with recombinant receptors have initiated a dissection of the complex pharmacology of the benzodiazepine receptors. However, the results with zolpidem and sar-

mazenil are probably of greater significance because they have demonstrated the existence of ligands that distinguish very effectively between receptor subtypes their affinities differ by 2-3 orders of magnitude (see Fig. 2). Such an order of differentiation is required to allow meaningful behavioural experiments to be conducted, and from which new areas of clinical application may become clear.

TiPS - Februa y 1992 [Vol. 231 Benzodiazepine receptor in vizlo The GABAA receptor oligomer isolated from the mammalian CNS has a molecular mass of about 250 kDa. By homology with the other ligand-gated ion channel receptors, fgr which experimental evidence is available, the GABA* receptor probably comprises five subunits, although no evidence is available concerning the stoichiometry of any naturally occurring receptor complex (see Box). Initial in situ hybridization studies mapped the distribution of the mRNAs encoding arl-ar3 in bovine brain=. It was suggested that the observed pattern of labelling was compatible with the receptor heterogeneity hypothesized from earlier radioligand binding data, with arl mRNA exhibiting the most ubiquitous distribution and representing the BZ1 receptor subtype. Recent studies show that mRNAs encoding each of the six (Y subunits exhibit quite distinct topographical distributions”*” (Fig. 1). It was not possible to delineate multiple labelling of individual cells. However, recent use of polyclonal antibodies raised to the cytoplasmic loop of the individual 01 subunits suggests that, within the rat brain, the majority of individual receptors contain only a single isoform of ar subunitz5. However, there is evidence accumulating that GABAA receptor oligomers do exist, albeit in relatively low concentrations, which may contain multiple (Ysub-it isoformsz6. The recombinant studies already conducted have generally used only a single (Y isoform, although expression studies in Xenopus oocytes have investigated the effect of multiple a-subunit isoform expressionz7. It is now important to define in detail the pharmacological signature associated with these recombinant receptors, and to iocate these specific signatures within the mammalian CNS. Correlation with behaviour The progress made’ in understanding the molecular basis of benzodiazepine receptor heterogeneity over the past decade has been considerable, but the functional consequences of this heterogeneity remain poorly understood. The original postulate, based on observations with

Fig. 2. Venn diagram retwasentina benzodikepine r&ognition-sire pbarmaoology of ru(l-tT)@2y2 recombinanf receptors. In box 1 all receptors have high affinity for muscimol and samwzenil. In box 2 all receptors have high aflinity for pzYq diazepam. In box 3 all receptors have low affinify for diazepam. In box 4, BZ, type receptors; high affinity for CL218872 and zolpidem. In box 5, BZ, type receptors; low affinity for CL218872 and zolpidem. Affiniiy for zolpidem and /3-CCE: box 4 > box 6 > box 7. Affinity for CL218872: box 4 > box 7 > box 6.

CL218872, was that compounds with selectivity towards the BZ1 subtype would be nonsedative anxiolytics; by extension, BZ2selective compounds might be nonanxiolytic sedatives2. However, this postulate has not been borne out by studies with a variety of recently synthesized chemical structures recognizing the benzodiazepine receptor. It is now increasingly difficult to correlate a dissociated behavioural profile (i.e. anxiolytic activity seen in the absence of sedative/myorelaxant effects) with BZJBZz selectivity. CL218872 was the first compound identified to discriminate between BZ1/BZ2 binding sites. In 1979, Lippa et aL6 reported that this compound was active in the water-lick conflict test for anxiolytic activity at doses some 50-fold lower than those needed to ,produce motor impairment in the rat. Subsequently, Gee et a1.2gdemonstrated that CL218872 antagonized the loss of righting reflex in the mouse induced by diazepam, suggesting that this compound may in fact be a partial agonist. However, a more detailed studyz9 revealed that the doses of CL218872 required to decrease spontaneous locomotor activity in rats and mice, to impair motor coordination (measured in a grasping test) in both species, and to potentiate ethanol narcosis in the mouse, were similar to those at which anxiolytic activity was observed. A ceiling effect was seen in the mouse grasping test, compatible with a partial agonist activity, but this was not seen in the rat. These results thus challenge

the view that CL218872 shows a dissociated behavioural profile in rodents. A number of other studies have also been published demonstrating the sedative effects of CL218872 (see Ref. 30). The other class of compounds that was originally shown to discriminate between BZ1 and BZ2 binding sites was the g-carbolines. Although the first generation, e.g. p-CCE, were shown to be inverse agonists at the benzodiazepine receptor, compounds were later developed that spanned the entire efficacy spectrum, from fulI agonists to inverse agonists31. @-Carboline full agonists, such as ZK93423, have both anxiolytic and sedative properties32 and the dose-ratio observed between the two effects is identical to that seen for classical benzodiazepines. Nevertheless, compounds do exist in the @carboline series that present a dissociated behavioural profile. These include ZK91296 (Ref. 33) and abecarnil%. However, these molecules have been demonstrated to antagonize the sedative effects of diazepam, and thus be partial agonists at the receptor, albenzodiazepine though other aspects of abecarnil’s pharmacology would be in keeping with a selective action at subpopulations of receptor. In particular, abecamil achieves its anxiolytic effects in rodent models at lower receptor occupancies than diazeparn=. Abecamil potentiates GABA-induced currents with a 100-fold higher potency in Xenopus ooqtes expressing ff1@2Y2 than those with ar3@2y2 receptors (D. Stephens, pers. commun.).

TiPS - February 1992 [Vof. 131

80 On the other hand, certain 1,4im~d~obe~odi~epin~, which do not &criminate between BZI and BZ2 binding sites, do show a dissociated behavioural profile, having very low activities in tests of sedation and muscle relaxation. These compounds include bretazeniP, RolT18lZ (Ref. 36) and FG8205 (Ref. 37). They appear to be partial agonists at the benzodimepine receptor, since they produce their anxiolytic and anticonvulsant effects at higher levels ofreceptoroccupancy than do full agonists, and they reverse the motor impairment induced by diazepam. Qua=pam, 2-oxoquarepam and cinolazepam axe 1,4benzodiazepines that show selectivity for the BZr receptor subtypess. Few data are available concerning the behavioural pharmtiogy of these impounds, although Ongini ef ai.% have shown that quazepam is less myorelaxant than flurazepam at equivalent sedative doses. The discovery of another class of BZr-selective ligands, the imidazopyridines, has fuelled the debate about the functional con-

sequences of receptor multiplicity. The first of these, zolpidem, has an unusual behavioural profile in that it has sedative activity (depression of spontaneous locomotor activity) at doses lower than those producing anticonvulsant, anxiolytic or myorelaxant effec@. Furthermore, whilst it has anticonflict properties in the waterlick conflict test, it is inactive in operant conditioning paradigms that are sensitive to benzodiazepine or @carboline anxiolytics. Likewise, alpidem is also inactive in operant conditioning models and shows an identical behavioural dissociation to that of zolpidem (i.e. sedative dose < ~ti~n~~t dose < anti-conflict (water-lick) dose < myorelaxant dose)41. Alpidem is less myorelaxant than zolpidem, and taken with its low efficacy in anticonvulsant models, may imply that alpidem is a partial agonist. However, the sedative effects of alpidem are difficult to reconcile with a partial agonist activity, and it is possible that these may be mediated by a mechanism other than the benzodiazepine receptor.

A variety of compounds of disparate chemical structure also display anxiolytic and anticonvulsant properties without producing sedation and muscle relaxation, induding the pyrazoloquinolines CGS9896 {Ref. 42) and CGS20625 (Ref. 43), the phenylquinoline pipequaline”, the imidazopyrimidine divaplon4’ and the cyclopyrrolone RR59037 (Ref. 46). These compounds have all been claimed to be partial agonists. Receptorbinding studies have demonstrated that none of them show selectivity for BZ1 over BZ2 binding sites. Pipequaline is unusual in that it is active in the water-lick conftict test but not in operant conditioning models; in this respect it resembles alpidem and zolpidem. BZ,/RZ, selectivity vs partial ago&m? With the hindsight afforded by the numerous benzodiazepine receptor ligands synthesized over the past ten years, it seems that .the original hypothesis that BZrselective compounds would be ‘anxioselective’ is not tenable.

TiPS - Februay 1992 [Vol. 131 First, the initial study reporting the lack of sedation with the B&selective CL218872 has not been confirmed. Secondly, other BZ1preferring molecules have variously been found to be nonsedative anxiolytics (abecamil), potent hypnotics (zolpidem) and. compounds whose behavioural profile is indistinguishable from diazepam (ZK93423). On the other hand, all the nonsedative anxiolytics described so far, whether they discriminate between BZ1 and BZz sites or not, appear to be partial agonists, and this seems to be a more parsimonious hypothesis by which to explain the observed dissociated behavioural profiles (see Ref. 47). Likewise, the proposal that BZ1selective full a onists might be ‘hypnoselective’ Go also seems implausible, since BZI-selective molecules other than the imidazopyridines show equal sedative and anxiolytic/anticonvulsant activities (e.g. CL218872, quazepam, ZK93423). However, it may be that the lower incidence of muscle relaxation seen with BZ1-preferring full agonists, such as quazepam and zolpidem (but not ZK93423), may be related to receptor subtype selectivity. q

0

0

A decade ago there was convincing evidence of benzodiazepine receptor heterogeneity, though few were persuaded that this explained the differences in pharmacodynamic profile of the compounds that recognized these sites in the mammalian CNS. Molecular cloning studies have now defined this heterogeneity, although it seems that the restricted pharmacodynamic profiles seen may be explained more simply by partial agonism. The physiological significance of the differential localization of the six a: subunits, demonstrated by in situ hybridization, has yet to be clarified. incorporation of their gene products to form receptor oligomers, each with a distinct pharmacology, is apparent from both neurochemical and electrophysiological studies, although the distribution and even the existence of these receptor oligomers in the mammalian CNS has yet to be elucidated.

81 The consequences of this disparate pharmacology on the integrated behaviour of the whole animal cannot be investigated with currently available ligands, which show a limited specificity for the receptor subtypes. The results with both zolpidem and sarmazenil demonstrate that the appropriate differential specificity can be achieved. Exploitation of this heterogeneity is therefore possible, and an extension to the other receptor subtypes will undoubtedly follow. Once again the medicinal chemist must come to the rescue; now the fruits of their imagination can be traced with facility thanks to the expedience of molecular biology. The availability of more specific and efficacious medicines is a tantalizing goal. References 1 Nielsen. M. and Braestruo. C. 11980) Nature i86.606-607 a_ 2 Liuva, A. S., Meverson, L. R. and Beer, B.*(i982) Life Sci: 31,1409-1417 3 Squires, R. F. et al. (1979) Pharmacol. Biochem. Behav. 10.825-830 4 Braestrup, C. ana Nielsen, M. (1980) Trends Neurosci. 3,301-303 5 Potier. M. C.. Prado de CaNaIho. L.. Dodd; R. i., Besselievre,’ R. -and Rossier, J. (1988) Mol. Pharmacol. 34, 124-128 6 Lippa, A. S. et al. (1979) Phartnacol. Biochem. Behav. 10,831-843 7 Sieghart, W. and Karobath, M. (1980) Nature 286, 2&287 8 Liiddens, H. and Wisden, W. (1991) Trends Pharmacol. Sci. 12,49-51 9 Fuchs, K., Miihler, H. and Sieghart, W. (1988) Neurosci. Left. 90,314-319 10 Fuchs, K. and Sieghart, W. (1989) Neurosci. Lett. 97, 329-333 11 Kirkness, E. F. and Turner, A. J. (1988) Biochem. J. 256.291-294 12 Stephen&, F. A., Duggan, M. J. and Casalotti, S. 0. (1989) FEBS Lett. 243, 358-362 13 BuchstaIIer. A.. Fuchs, K. and SieP;hart, W. (1991) ieuiosci. Le&. 129, 237-541 14 Pritchett, D. B.. Liiddens, H. and Seeburg; P. H.. (1989) Science 245, 1389-1392 15 Pritchett, D. B. and Seeburg, P. H. (1991) Proc. Nat1 Acad. Sci. USA 88, 1421-1425 16 Pritchett, D. B. and Seeburg, P. H. (1990) 1. Neurochem. 54,1802-1804 17 Liiddens, H. et al. (1990) Nuture 346, 648-651 18 Sieghart, W., Eichinger, A., Richards, J. G. and Miihler, H. (1987) J. Neurothem. 48,46-52 19 Malmieni, 0. and Korpi, E. R. (1989) Eur. J. Pharmacol. 169, 53-60 20 Ticku, M. K. and Kulkami, S. K. (1988) Pharmacof. Biochem. Behav. 30,501-510 21 Uusi-Oukari, M. and Korpi, E. R. (1990) 1. Neurochem. 54,1980-1987 22 hisden, W., Morris, B. J., Darlison, M. G., Hunt, S. P. and Barnard, E. A.

(1988) Neuron 1.937-947 23 kisden, W., Laurie, D. J., Monyer, H. and Seeburp;. P. H. 1. Nwosci. fii me4 24 Laurie, E. J., S&burg, P. H: a& W&den, W. J. Neurosci. (in press) 25 McKeman, R. M. et al. Neuron (in press) 26 Duggan. M. J., Pollard, S. and Stephenson, F. A. J. Biol. Chem. (in 27 Sigel, E., Bauer, R, Trube, G., M&Ier, H. and MaIherbe, P. (1990) Neuron 5, 703-711 28 Gee, K. W., Brinton, R. E. and Yamamura, H. I. (1983) Life Sci. 32, 1037-1040 29 OakIey, N. R., Jones, B. J. and Straughan, D. W. (1984) Neurmhurmucology23, 797-802 * 30 Gardner, C. R. (1988) Drug Dev. Res. l2, l-28 31 Stephens, D. N. et al. (1987) Brain Res. Bull. 19,309-318 32 Stephens, D. N., Kehr, W., Wachtel, I-L and Schmiechen, R. (1985) Pharmacopsych&y 18,167-170 33 Petersen. E. N. et al. f1984) Psuchophannac~logy 83,240-2& ’ 34 Stephens. D. N. et al. (1990) 1. Phamzacol.- Ezp. tier. 253,334&3. . 35 Martin, J. R. et al. (1988) Phnrmacopsychiatry 21,360-362 36 Haefely, W. (1984) Clin. Neurophnrmacol. 7, s363 37 TrickIebank, M. D. et al. (1990) Br. J. Phanaacol. 101,75>761 38 Sieghart, W. and Schuster, A. M. (1984) Biochem. Pharmacol. 33,4033-4038 39 Ongini, E. ef al. (1982) Arzneim.-Forsch. 32,1456-1462 40 Depoortere, H. et al. (1986) J. Phurmacol. Exp. Ther. 237; 64=8 41 Ztvkovic, 8. et al. (1990) Phnrmncopsychiatry 23,108-113 42 Petrack, B., Czemik, A. J., Cassidy, J. P., Bernard, P. and Yokoyama, N. (1983) Adv. Biochem. Psychopharmacol. 38, 129-137 43 Williams, M. et al. (1989) J. Pharmacol. Exp. Tker. 248,89-96 44 Le Fur, G. et al. (1979) Life Sci. 28, 1439-1448 45 Gardner, C. R. et al. (1987) Br. J. Phamzacol. 92,537P 46 Piot, 0. et al. (1990) Br. J. Pharmncol. 99, 133P 47 Haefelf, W., Martin,J. R. and Schoch, P. (1990) Trends Phanacol. Sci. 11,452-456

fl-CCE: fi-carboline-3-carboxylate ethyl ester CGS9896: 2-(4chIorophenyl)-2,5-dihydropyrazolo[43-clqutnoline-3(3H)-one CGS20625: 2-(4-methoxyphenyl)2,3,5,6,7,8,9,10-octahydm cycIohepta[b]pyrazolo[3,4-dpyridin-3-one CL218872 3-methyl-6-[3-trifluoromethyluhenvll-1.2.4~triazolo-14.3-b]pyrIdazine &2&: 7&oro-5,6-dihyd;-S-methyl6-oxo-3-(5-isopropyI-l,2,4-oxadiazol-3-yI)4H-imid&o[l,>n]ii,4]benzodiazepine Rol7l812z cyclopropyhnethyl (s)d-chloro12,12a-dihydro-9-oxo-9H,llH-aceto[2,7cl-imidazo[l,S-a][1,4]benzodiazepine-lcarboxylate RP59032 2-(7-chIoro-2-naphtyridin-l&yl) 3-(S-methyl-2-oxohexyl)isoindoin-l-one ZK91926: ethyl 5-benzyloxy-4methoxymethyl-p-carboline-3-carboxylate ZK93423: ethyl 6-benzyloxy-4methoxymethyl-@-carboline-3-carboxyiate