Benzodiazepine receptor heterogeneity: Neurophysiological implications

Vol. 34, No. 3, pp. 245-254,1995 Copyright0 1995ElsevierScienceLtd Printedin Great Britain.All rightsreserved 002%3908/95 $29.00+ 0.00

Neurophormacology

0028-3908(94)0015&8

Review: Neurotransmitter Receptors III

GABA*/Benzodiazepine Receptor Heterogeneity: Neurophysiological Implications H. LijDDENS,‘*

E. R. KORPI’ and P. H. SEEBURG’

‘Center for Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany and ZBiomedical Research Center, Alko Ltd. Helsinki, Finland (Accepted I5 November 1994) Keywords-GABA,

GABA* receptor, benzodiazepine

y-Aminobutyric acid (GABA), the most prevalent inhibitory neurotransmitter in the mammalian brain, exerts its main action through GABA, receptors (GABAARs). These belong to the superfamily of ligand-gated ion channels and respond to the presence of GABA by the opening of an intrinsic anion-channel. Multiple GABAARs exist in the brain and show differential expression patterns. The receptors have a pentameric structure formed from members of at least three different subunit families (a 1-6, /31-3 and y l-3). The regulation of functional properties by GABA and its analogs and by benzodi.azepine (BZ) receptor ligands and neurosteroids differs d.ramatically with the type of a variant in the complex. Additional variation of GABAARs results from the substitution of y subunits. No clear picture exists for the role of the /? subunits which are essential for receptor assembly. The effect of BZ receptor ligands on animal behavior range from agonist effects (e.g. anxiolysis, sedation and hypnosis) to inverse agonist effects (e.g. anxiety, alertness and convulsions). The diversity of effects reflects the ubiquitous expression of GABA,/BZ receptors in the brain. Recent data provide insight into the mechanism of action of BZ ligands, but correlations from a single ligand to a single behavioral effect cannot be made. This may result from the fact that intrinsic efficacies of ligands differ between GABA,R subtypes, and thus the diversity of native receptors is further compounded by the modes of action of the ligands affecting GABAAR subtypes. Ligand-gated ion channels mediate fast excitatory and inhibitory synaptic n’eurotransmission in the CNS (Dingledine et al., 1988). The family of ligand-gated ion channels now comprises nicotinic acetylcholine receptors

*To whom correspondence

should be addressed.

receptor.

(nAChR), y-aminobutyric acid type A receptors (GABA*R), glycine receptors (GlyR), the serotonin 5-HT, receptor (Betz, 1990; Maricq et al., 1991; Schofield et al., 1987), which all form by the assembly of five subunits (Langosch et al., 1988; Nayeem et al., 1994; Unwin, 1993), and the structurally distinct glutamate receptor channels (Monyer et al., 1992; Moriyoshi et al., 1991; Roche et al., 1994). The channels are either homo-oligomers (5-HTs) or hetero-oligomers of up to four different subunits (peripheral nAChR). Each subunit of a ligand-gated receptor channel spans the plasma membrane four times (Unwin, 1989). The second transmembrane region (TM2) of each subunit arranges around a central channel pore and thus provides the environment essential for the selectivity and passage of ions (Unwin, 1989). GABA*Rs share a high degree of sequence similarity with other ligand-gated ion channels (Betz, 1990). Indeed, three amino acid changes of the primary nAChR sequence in TM2 convert cation-selective channels into anion-selective channels, both being responsive to acetylcholine (Galzi et al., 1992). As GABA transmits the majority of all inhibitory fast signaling in the mammalian brain, an obvious question is how a single receptor type can specifically regulate brain function, given the diverse neuronal circuitries in the various brain areas.

SUBUNIT HETEROGENEITY OF GABAIRS

are multimembered. Five subunits assemble into a functional complex and this provides a means to build an array of different receptors from a limited number of proteins. Early pharmacological and biochemical data furnished clues to the existence of GABA,R subtypes, but the full diversity was not anticipated until the cloning of, now 15, different receptor GABAARs

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constituents from mammalian CNS (Liiddens and Wisden, 1991; Seeburg et al., 1990). The subunits are grouped into five classes according to the degree of amino acid identity with one to six variants each (a 16, B 1-3, ~1-3, 6 and ~1-2). Two additional subunits have been identified in chick brain (84, ~4) (Bateson et al., 1991; Harvey et al., 1993). Whereas the classes u to 6 constitute classical GABA*Rs, proof for membership is missing for the p class. Subunits pl and p2, originally found in the human retina (Cutting et al., 1991, 1992), form homomeric picrotoxinin-sensitive GABA-gated Cl channels, but their pharmacology sets them apart from GABAAR as they are insensitive to bicuculline, a specific GABAAR antagonist (Kusama et al., 1993a,b). Unresponsiveness to baclofen excludes p 1 and p2 from the G-protein coupled GABAB receptors; it has been suggested that these polypeptides should be classified as GABAc receptors (Shimada et al., 1992). Five GABAAR subunits exist as splice variants (Bateson et al., 1991; Harvey et al., 1994; Kofuji et al., 1991; Korpi et al., 1994; Whiting et al., 1990). The best studied example is the y2 long form (y2L) which, when compared to the y2S variant, contains an eight amino acid insert between TM3 and TM4. These amino acids add a potential phosphorylation site that had been claimed to be involved in the ethanol sensitivity of GABAARs expressed in Xenopus oocytes (Wafford et al., 1991). The chicken 82 and 84 subunit variants differ, respectively, by 5 1 and 12 bp long inserts in the intracellular loop between TM3 and TM4 (Bateson et al., 1991; Harvey et al., 1994). Two rodent a6 forms differ in the N-terminal extracellular region (Korpi et al., 1994). The short LYE form fails to form functional receptors. A human 83 variant has an alternative exon 1, which encodes a variant signal peptide (Kirkness and Fraser, 1993). Without the diversity contributed by splice variants and by the p subunits, 60 GABAAR combinations of the putative form ax&xyx2 (Backus et al., 1993) or ctx& may exist in the mammalian brain. The possibility of different variants or splice variants of a single subunit class in a receptor, e.g. axay/?xyxyy (Khan et al., 1994; Korpi and Liiddens, 1993; Liiddens et al., 1991; Mertens et al., 1993; Quirk et al., 1994a) or other subunit stoichiometries, e.g. ax3@x2, further increases the theoretical number. However, the formation of native receptors may be restricted by incompatibility of assembly and by the select subunit expression in central neurons. The mRNAs encoding the a 1-6, pl-3, y l-3 and 6 subunits display a unique distribution in the mammalian CNS. Some neuronal populations, such as the dentate granule cells, contain virtually all GABAAR mRNAs (Laurie et al., 1992; Wisden et al., 1992). Other cells, such as the Purkinje cells of the cerebellum, express a limited number of GABAAR mRNAs (Laurie et al., 1992; Shivers et al., 1989). The a 1 mRNA encodes the most prevalent and ubiquitous GABAAR subunit in the rodent brain and is widely coexpressed with the p2 mRNA (Khrestchatisky et al., 1989; Laurie et al., 1992; Malherbe et al., 1990b;

Sequier et al., 1988; Wisden et al., 1992). The y2 variant mRNA often colocalizes with a 1 and 82 mRNA (Laurie et al., 1992; Malherbe et al., 1990b; Shivers et al., 1989; Wisden et al., 1992). This triple combination constitutes the vast majority of GABA,R mRNA expression in many cell populations. Combinations such as cr2/?1(/.?3)and a5/?1@3) are the most abundant GABAAR species in the hippocampus (Laurie et al., 1992; Wisden et al., 1992). At present, no subunit combination can be ignored, since even a rare receptor may have important functions for select neuronal populations, an example being the a6 variant which is only expressed in cerebellar granule cells (Ltiddens et al., 1990). The intracellular location of a subunit mRNA in a neuron may drastically differ from where the subunit inserts into the plasma membrane. However, for a number of GABAAR subunits and in most parts of the brain, GABAAR protein and mRNA distribution patterns overlap (Benke et al., 1991a; Khan et al., 1993; Laurie et al., 1992; Shivers et al., 1989; Wisden et al., 1992; Zimprich et al., 1991) with only few exceptions. One exception is y2 (long and short splice forms) in the hippocampus, which shows prominent mRNA signals but low immunoreactivity (Benke et al., 199 1a; Malherbe et al., 1990b; Shivers et al., 1989; Wisden et al., 1992). BZ RECOGNITION SITE ON GABAaRS The amino acid identity between the four GABA,R subunit classes reflects functional similarities, e.g. a2 or a3 can substitute for u5 in many respects. Coexpression of the al and Bl subunits in Xenopus laevis oocytes results in GABA-activated Cl - channels (Schofield et al., 1987). Furthermore, the a2, cr3 and a5 variants coexpressed in X. laevis oocytes with /?l and a 1 together with pl, 82 or 83 all assemble into GABA-gated ion channels (Levitan et al., 1988; Ymer et al., 1989a,b). The exchange of one c( variant for another in an clxpl combination shifts the dose-response curve for GABA, but does not affect the overall pharmacological properties of the resulting channels (Levitan et al., 1988). These heteromeric, recombinant GABA channels are modulated positively by pentobarbital and blocked by bicuculline and the channel blocker picrotoxinin. However, the GABA response of X. laevis oocytes expressing CL 1/I 1 subunits is not modified by BZs (Malherbe et al., 1990a; Schofield et al., 1987). Whereas earlier reports (Sigel et al., 1990) stressed the insensitivity of all double combinations (axpx, axyx or pxyx) to BZ ligands, recent reports indicated that certain ligands, e.g. diazepam, require the presence of only the y2 together with either an CIor a B variant to be potentiated by the BZ (Im et al., 1993). However, the two component receptors lack the high-affinity BZ recognition site seen in mammalian brain (Pritchett et al., 1989). When the y2 subunit is coexpressed with an a and a p variant, GABA_, channels form which consistently respond to BZ ligands, i.e. the receptors resemble native

GABAA/benzodiazepine receptor heterogeneity GABA.JBZ receptors (Pritchett et al., 1989). Most BZs bind to GABA.JBZ receptors with similar affinities throughout the brain, but the binding properties of several compounds, most notably Cl 218,872 (Nielsen and Braestrup, 1980; Sieghart, 1983; Squires et al., 1979) and 2-oxo-quazepam (Corda et al., 1988; Olsen and Tobin, 1990), demonstrate the heterogeneity of GABAJBZ receptors (Fig. 1). Type I receptors have greater affinity to the triazolopyridazine Cl 218,872 and the 1,4-benzodiazepine 2-oxo-quazepam than type II receptors, and constitute the predominant GABA,R class in the CNS. The low-affinity type II receptors are enriched in hippocampus, striatum and spinal cord (Lo et al., 1983; Olsen and Tobin, 1990; Sieghart et al., 1985). Coexpression of the a 1, /?1 and y2 subunits in cultured 293 cells leads to the assembly of functional GABAARs that display the characteristics of BZ type I receptors (Pritchett et al., 1989). Exchange of bl by other p variants does not alter the affinity of BZ ligands to the receptor (Hadingham et al., 1993; Pritchett et al., 1989). However, replacing the CI1 subunit by other a variants dramatically affects the affinity of the formed GABA*/BZ receptor complex to select central BZ receptor ligands (Hadingham et al., 1993; Pritchett et al., 1989). The pharmacological and electrophysiological properties of GABA*/BZ type I receptors can be mimicked only by receptors of the u lfixy2 type. BZ II receptors assemble

from cr2, ct3 or a5 subunit variants together with the /?xy2 combination (Pritchett et al., 1989; Pritchett and Seeburg, 1990). Both a2fixy2 and 013/?xy2 receptors display the characteristics of the “classical” BZ type II receptors. u5jI2y2 receptors have similarly low affinities for Cl 218,872 and 2-oxo-quazepam as do ~2pxy2 and cr3/3xy2 receptors. However, the binding properties for imidazopyridines such as zolpidem and alpidem differ from conventional GABAA/BZ II receptors (Pritchett and Seeburg, 1990). BZ I and BZ II receptors distribute unevenly in the rat brain. BZ I receptors predominate in the cerebellum but are scarce in the hippocampus (Faull and Villiger, 1988; Faull et al., 1987; Olsen et al., 1990). On the other hand, BZ II receptors are strongly expressed in the hippocampus and nearly absent from the cerebellum, whereas both receptor classes are equally expressed in cortical layers (Faull and Villiger, 1988; Faull et al., 1987; Olsen et al., 1990). Data derived from ligand binding to brain membranes or brain slices are in good agreement with the mRNA distribution of the CIsubunits (see above). These data indicate that recombinant ctlj?xy2, cr2/?xy2, c13Bxy2 or a5p2y2 GABAA/BZ receptors are similar to their native counterparts. This view is supported by immunopurification of GABAAR subtypes solubilized from rat brain. These subtypes display affinities comparable to those measured in recombinantly

GABAA Receptors

non-BZ ReceDtors

diazepam-sensitive

/ zolpidem-sensitive

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\ zolpidem-insensitive

Fig. 1. “Family tree” of GABA*/BZ receptors. BZ receptors are shown as a subset of GABAARs which can be differentiated according to their BZ ligand specificity. Diazepam insensitivity is the first criterion used which singles out ct6Bxy2 receptors. Recombinant a4/?2y2 receptors are diazepam-insensitive as well (Wisden et al., 1991) but thalamic nuclei rich in the a4 message (Wisden et al., 1992) lack a corresponding amount of diazepam-insensitive BZ receptors (Sieghart et al., 1987). The other subsets are the zolpidem-insensitive and zolpidem-sensitive receptors, which can be further classified solely on their affinity to Cl 218, 872. Note, that medium affinity for Cl 218, 872 corresponds to the high affinity formerly attributed to BZ type I receptors. For further details, see the text.

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expressed receptors (Duggan and Stephenson, 1990; Marksitzer et al., 1993; McKernan et al., 1991; Mertens et al., 1993). The a6 variant has striking features. It is the subunit with the most restricted distribution, limited to cerebellar granule cells (Liiddens et al., 1990). Recombinant a6/?2y2 receptors bind with high affinity the GABA agonist [3H]muscimol and the imidazo- 1,Cbenzodiazepine [3H]Ro 15-45 13. Other BZs and two /?-carbolines are not recognized by this receptor or have an affinity of two to three orders of magnitude lower than that of cIl-, a2-, u3or a5-containing receptors (Liiddens et al., 1990; Pritchett et al., 1989; Pritchett and Seeburg, 1990). Several BZ receptor full agonists displace all [3H]Ro 15-45 13 binding from cerebellar membranes in a two-step manner. The affinities of these ligands in native membranes correspond well to those obtained from recombinant receptors: in alcohol-tolerant and non-tolerant rat lines (Uusi-Oukari and Korpi, 1990) and in membranes derived from rat, bovine or human cerebellum (Liiddens et al., 1990; Turner et al., 1991; Wong and Skolnick, 1992) inhibition of [3H]Ro 15-45 13 binding is best fitted to two-site curves in which the low-affinity site is present to 20-30% and most likely represents a6-containing receptors. a6 may form GABA,,Rs together with the 6 subunit (Caruncho and Costa, 1994), but whether these receptors recognize BZs is still open to debate (Benke et al., 1991b; Quirk et al., 1994b). Coexpression of the crl, 82 and y3 subunits in 293 cells generates GABAARs which recognize BZ ligands with reduced affinities for agonist BZ ligands when compared to BZ antagonists or inverse agonists (Herb et al., 1992; Knoflach et al., 1991). The y3 variant confers zolpidem-insensitivity to crlfi3y3 and ~583~3 receptors accompanied by a low nanomolar affinity for Cl 218,872 (Liiddens et al., 1994). Replacement of y2 by the yl subunit in an ax/?xyx combination (Ymer et al., 1990) results in BZ receptors with a pharmacology superficially reminiscent of the peripheral type BZ acceptor site (Lueddens and Skolnick, 1987). However, the action of central BZ receptor ligands in y 1-containing receptors provides evidence that they form GABA,/BZ receptors. They are characterized by an agonist action of the otherwise inverse agonists Ro 15-4513 and dimethoxy-4-ethyl-/?-carboline-3-carboxylate on a 1B1y 1 and cr2/?lyl receptors (Puia et al., 1991; Wafford et al., 1993a). Collectively, these data indicate the existence of GABA*/BZ receptors with pharmacological profiles for BZ receptor ligands profoundly different from the hitherto described classical and-probably most abundant-BZ recognizing receptors. An overview of some known recombinant GABAA/BZ receptors, excluding y 1 receptors, is given in Fig. 1, where they are grouped according to their characteristic BZ binding profile. It is important to note that the term medium affinity for Cl 218,872 used in the figure corresponds to the high affinity formerly ascribed to-BZ type I receptors, whereas the Cl 218,872 high-affinity receptors contain the

y3 subunit and do not fall into the BZ I or BZ II categories. Molecular biological techniques provide powerful tools for examining the structural requirements of any property of a protein. By exchanging either single amino acids in a protein sequence or by exchanging larger domains between closely related proteins, the amino acid(s) determining a functional property can be evaluated. This approach has been applied also to GABA*/BZ receptors. By exchanging ever smaller regions of the cr3subunit sequence with the corresponding portion of the c(1 subunit, a single substitution (glycine for glutamic acid) in the N-terminal extracellular domain leads to an increase in the affinity for the subtype-selective compounds Cl 218,872 and 2-oxo-quazepam. This increase is approximately lo-fold and does not involve the affinity for non-selective compounds such as diazepam (Pritchett and Seeburg, 1991). Hence, the BZ II type recombinant receptor a3/?2y2 can be converted into a BZ I type receptor by a point mutation. Constructing N-terminal chimeras between the a 1 and 016variants, a single arginine’O”to histidinelm substitution in ~16imparts sensitivity of 016B2y2receptors to diazepam (Wieland et al., 1992). Furthermore, diazepam insensitivity can be conferred to ctl receptors by replacing the corresponding histidine’O’ with an arginine (Wieland et al., 1992). The point mutation only changes the affinity for diazepam but does not interfere with affinity for GABA (Kleingoor et al., 1993). The importance of the histidine residue for BZ agonist binding is highlighted by the finding that all diazepam-sensitive receptors, i.e. al(cr2, a3, ct5)/?2y2, contain a histidine at the corresponding position, whereas this histidine is replaced by an arginine in the diazepam-insensitive receptor types a4(a6)/?2y2. Recently, a natural point mutation in cr6 (arginine Ionto glutamine’“) was shown to occur in the alcohol-sensitive ANT rat line (Korpi et al., 1993). Glutamine’OO induces an increase in the affinity for diazepam when compared to wild-type cr6, without fully reaching the high affinity of 0:1, a3 or the mutant a6histidine’” BZ receptors. In this example substitution in a critical amino acid position of multisubunit proteins is assumed to affect the pharmacological sensitivity of animal behavior. The involvement of mutant ~16receptors in the abnormal BZ sensitivity is supported by the lack of motor impairment in ANT rats by zolpidem, a compound that neither binds to wild-type nor to mutant cr6receptors (Korpi and Sarviharju, 1993). RECEPTOR SUBTYPES AND BEHAVIOR The effects of central BZ receptor ligands on animal behavior range from agonist effects, e.g. anxiolysis, sedation and hypnosis, to inverse agonist effects, e.g. anxiety, alertness and convulsions [for recent pharmacological reviews, see Doble and Martin (1992), Gardner et al. (1993) and Nutt (1989)]. BZs such as flunitrazepam and diazepam produce anxiolytic, anticonvulsant,

GABAA/benzodiazepine receptor heterogeneity sedative and myorelaxant effects at partially overlapping dose ranges. But not all substances acting on the BZ site of GABAA/BZ receptors belong to the same chemical class, nor do they produce all the aforementioned effects. Two prime examples of agonistic ligands with distinct behavioral profiles are the triazolopyridazine Cl 218,872 and the imidazopyridine zolpidem. Cl 218,872 produces anxiolytic responses w:ith a low tendency to sedation, whereas zolpidem has a lstrong sedative action and the low doses necessary show only weak anxiolytic effects. Both compounds are BZ I receptor preferring, with zolpidem being more subtype selective (Pritchett et al., 1989; Pritchett and Seeburg, 1990). Therefore novel ligands with stricter subtype-specific actions are required before assigning any behavioral BZ effect to a specific GABAAR subtype. This is also true for a&containing receptors in cerebellar granule cells, which may be associated with the motor-impairing effects of alcohol (Korpi and Seeburg, 1993; Liiddens et al., 1990). To test the latter hypothesis, a ligand affecting only this receptor subtype is needed. Beside GABA,R subtype selectivity, the behavioral correlate of BZ treatment is determined by the intrinsic efficacy of the ligand. Earlier theories for partial agonism were based on different receptor reserves for various behaviors (Haefely, 1988; Haefely et al., 1992). This theory posited that a full agonist produces all its effects already at a relatively low receptor occupancy. In contrast, a partial agonist, even with its limited action profile, needs high receptor occupancy and would still not produce all the behavioral effects of a full agonist. This theory explained the differences in behavioral profiles between diazepam, zolpidem and Cl 218,872: diazepam is a full agonist at many receptor subtypes, zolpidem a full agonist at a limited nu:mber of subtypes (Biggio et al., 1989; Liiddens et al., 19’94;Wafford et al., 1993b) and Cl 2 18,872 is a partial agomst at all receptor subtypes studied (Liiddens et al., 1994; Wafford et al., 1993b; Yakushiji et al., 1993). However, this explanation does not suffice to assign various behaviors to the action of a defined receptor subtype, since recent studies indicate that some compounds are partial
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behavioral profiles of ligands with their intrinsic activity and subtype-specificity. CONWLSANT BINDING SITE GABA and all substances interacting with GABAARs modulate the convulsant binding site labeled by [35S]t-butylbicyclophophorothionate ([35S]TBPS). Convulsants such as TBPS or picrotoxin(in) may bind to a modulatory site on GABAARs or to the channel pore. Picrotoxin appears to block GABA responses by two mechanisms, neither one involving the binding of the convulsant to the open channel pore, but only one of them is use-dependent (van Renterghem et al., 1987; Yoon et al., 1993). The modulation of [35S]TBPSbinding by GABA and its analogs varies between different brain regions. In rat brain sections, the main sensitivity difference to GABA between brain regions was observed in the cerebellum (Korpi et al., 1992; Peris et al., 1991; Ticku and Ramanjaneyulu, 1984). [?S]TBPS binding in the granule cell layer was more sensitive to GABA than that in the molecular layer, and was detected only after blockade of the GABA agonist sites by the specific GABA* antagonists SR 9553 1, RU 5 135 or bicuculline (Korpi and Liiddens, 1993). This indicates that the r5S]TBPS binding sites in cerebellar granule cells were blocked by remaining endogenous GABA. Expression in 293 cells of a6/?2y2 receptors produced [35S]TBPS binding sites that were lo-fold more sensitive to inhibition by GABA than in crl/?2y2 receptors (Korpi and Liiddens, 1993). Similar results were obtained for [35S]TBPS binding to alfl2y2 and 1~68272 receptors modulated by the neurosteroid 5a-pregnan-3a-ol-20-one (Korpi and Liiddens, 1993). These data indicate that the c1variants do not only provide differential sensitivity to BZ ligands but also to the neurotransmitter itself and to neurosteroids. After it was recognized that r5S]TBPS binds to GABAARs (Squires et al., 1983), this cage convulsant was used as a marker for the activity of BZ receptor ligands (Supavilai and Karobath, 1983, 1984). Agonists in the virtual absence of GABA increase baseline levels of [3SS]TBPSbinding, and antagonists decrease it (Supavilai and Karobath, 1983). The effects of both groups of compounds could be blocked by the BZ site antagonist Ro 15-1788 (flumazenil) (Sannaet al., 1991; Supavilai and Karobath, 1983). GABA analogs reverse the direction of interaction of BZ ligands with the [35S]TBPS binding. Zolpidem decreased instead of increased [35S]TBPS binding (Biggio et al., 1989; Liiddens et al., 1994; Serra et al., 1992). Again, antagonists produce an effect opposite to agonists (Stephens and Kehr, 1985). Furthermore, the dose-response curve for the BZ ligands is shifted to lower concentrations (Gee et al., 1986; Supavilai and Karobath, 1984), so that they closely reflect their in viuo potency (Stephens et al., 1987). Together these data indicate that [35S]TBPSbinding is a reliable tool to investigate compounds for intrinsic activities. In fact,

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[35S]TBPSbinding ex vivo has been used to assess the effect of stress and other environmental factors (Concas et al., 1988; Trullas et al., 1987).

OUTLOOK

The spatial organization of synaptic inhibitory input is highly complex as is the molecular composition of individual GABAARs. A single cell can inhibit a number of neurons and many inhibitory cells can converge on a single neuron. Specified brain regions, e.g. the CA1 and CA3 regions of the hippocampus, express nearly all known GABA,+R subunits (Persohn et al., 1991; Wisden et al., 1992), though within a region not all cells express all subunits of a class. An example is the ~11variant, which can only be detected in a subset of hippocampal interneurons (Gao and Fritschy, 1994). At present, the impact of receptor diversity on the local circuitry is unknown. Inhibitory neurons can innervate different domains of the postsynaptic neuron, i.e. soma, dendrite and axon (Baude et al., 1992; Buhl et al., 1994; Somogyi et al., 1989). Therefore, the subcellular distribution of individual subunits differs. For example, cr6 subunit immunoreactivity is found only in the dendrites of cerebellar granule cells, whereas the ~1 variant can be detected in dendrites and in the soma of the same cells (Baude et al., 1992). On the other hand, different patterns of spontaneous and GABA-evoked inhibitory postsynaptic potentials can be detected both in the hippocampus and cerebellum (Pearce, 1993; Puia et al., 1994), but the data do not yet allow us to correlate the molecular and functional diversity. The greatest challenge for future research is to establish roles for different GABAAR subtypes at physiological and, finally, at behavioral levels. This requires a combined effort of pharmacology (subtype-specific probes), physiology (characteristics of native receptor channels and neuronal circuits) and anatomy (cellular and subcellular localization of subunits) together with the application of novel molecular biological techniques, such as the development of transgenetic animals with altered GABAergic circuitry.

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