GABA receptor mechanisms in the central nervous system.

Progress in NeurobiologyVol. 36, pp. 35 to 92, 1991 Printed in Great Britain. All rights reserved

0301-0082/91/$0.00 + 0.50 © 1990 Pergamon Press plc

GABA RECEPTOR MECHANISMS IN THE CENTRAL NERVOUS SYSTEM LUCIA SIVILOTTI I and ANDREA NISTR1.2 Department of Pharmacology, St. Bartholomew's Hospital Medical College, Charterhouae Square, London ECIM 6BQ, U.K.

(Received 1 May 1990)

CONTENTS Abbreviations 1. Introduction 2. Biochemical pharmacology of GABA^ receptors 2.1. Molecular biology of GABA^ receptor-channel complex 2.2. The GABAA receptor binding sites 2.2.1. GABA^ recognition sites 2.2.2. Benzodiazepine sites 2.2.3. Picrotoxin/cage convulsant sites 2.3. A functional assay of GABA^ receptor activity: 360- flux measurements 3. Electrophysiology of GABAA receptor mechanisms 3.1. Ionic selectivity of GABA^ receptor-activated channels 3.2. Conductance of the GABA^ receptor-gated ion channel 3.3. Kinetics of the GABA^ receptor-operated CI- channel 3.4. Characteristics of GABAA-activated CI- channels 3.4.1. Agonists 3.4.2. Antagonists 3.4.3. Modulators 4. GABAB receptors 4.1. Biochemical characteristics and autoradiographic distribution 4.2. Cellular mechanisms underlying responses mediated by GABA B receptors 5. Autoregulation of GABAergic transmission 6. An unusual GABA receptor in central visual pathways 7. GABAergic synaptic transmission 7,1. Hippocampus 7.1.1. GABAergic pathways 7.1.2. GABAergic synaptic potentials 7.2. Olfactory cortex and neocortex 7.2.1. GABAergic pathways 7.2.2. GABAergic synaptic )otentials of principal neurones 7.2.3. GABAerglc synaptic )otentials of interneurones 7.3. Olfactory bulb 7.3.1. GABAergic pathways 7.3.2. GABAerglc synaptic ~otentials 7.4. Septal nuclei 7.4.1. GABAergic pathways 7.4.2. GABAergtc synaptic potentials 7.5. Thalamus 7.5.1. GABAergic pathways 7.5.2. GABAerglc synaptic ~otentials 7.6. Striatum, globus pallidus and entopeduncular nucleus 7.6.1. GABAergic pathways 7.6.2. GABAerglc synaptic potentials 7.7. Substantia nigra 7.7.1. GABAergic pathways 7.7.2. GABAergic synaptic potentials 7.8. Nucleus aceumbens 7.8.1. GABAergic pathways 7.8.2. GABAergtc synaptic potentials

36 36 36 36 37 37 39 41 41 43 43 44 47 49 49 50 50 53 53 53 55 57 58 58 58 59 64 64 65 68 68 68 68 70 70 70 71 71 71 73 73 74 74 74 74 75 75 75

* Reprint requests to A.N. ~Present address: Department of Anatomy, University College London, London WCIE 6BT, U.K. :Present address: Department of Pharmacology, Queen Mary Westfield College, University of London, London E1 4NS, U.K. 35

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L. SIVILOTTIand A. NISTR! 7.9. Cerebellum 7.9.1. GABAergic pathways 7.9.2. GABAergic synaptic potentials in the cerebellar cortex 7.9.3. GABAergic synaptic potentials in deep cerebellar nuclei 7.10. Brainstem nuclei 7.10.1. Dorsal raphe 7.10.2. Locus coeruleus 7.11. Spinal cord 7.11.1 GABAergic pathways 7.11.2. GABAergic synaptic potentials Acknowledgements References

ABBREVIATIONS

Single channel conductance ATP Adenosine 5'-triphosphate 3APS 3-Aminopropanesulphonic acid &ax Maximal number of binding sites CNS Central nervous system cAMP Cyclic adenosine 5'-monophosphate DHP Dihydropicrotoxinin DMCM Methyl-6,7-dimethoxy-4-ethyl-fl-carboline- 3carboxylate Eci C1 equilibrium potential EIPSP 1PSP reversal potential EDs0 Dose producing a half-maximal response EPSP Excitatory postsynaptic potential GABA y-Aminobutyric acid GABA-T GABA transaminase GAD Glutamic acid decarboxylase GTP Guanosine 5'-triphosphate GTP-?-S Guanosine 5'-O-(3-thiotriphosphate) IC50 Dose producing a half-maximal inhibition IPSC Inhibitory postsynaptic current IPSP Inhibitory postsynaptic potential Kd Dissociation constant eA Permeability to APermeability to CITBPS t-Butylbicyclophosphorothionate TTX Tetrodotoxin

1. INTRODUCTION Nearly a dozen years after completing a survey on the inhibitory neurotransmitter GABA (~,-aminobutyric acid) and its receptors (Nistri and Constanti, 1979), the picture which emerges has changed considerably and has prompted the preparation of the present review article. In the last few years major advances have been obtained in the field of purification and reconstitution of GABA receptors, in the characterization of the ionic channels gated by this amino acid and in the pharmacological classification of its different receptor types. All these topics will be considered here but pressures of time and space will allow only a discussion limited to results from neurones of the vertebrate central nervous system with emphasis on the synaptic actions of GABA in various areas of the mammalian brain and spinal cord. This approach will therefore exclude the more integrated phenomena (behavioural changes, homeostatic mechanisms, etc.) which may be regulated by GABAergic systems (information on these issues is available in a recent review; Matsumoto, 1989). The first part of the present survey will examine some general properties of GABA receptors as subdivided into two main

75 75 76 76 77 77 77 77 77 78 79 79

categories, termed GABA A and GABAs on the basis of their pharmacological characteristics (Bormann, 1988).

2. BIOCHEMICAL PHARMACOLOGY OF GABA A RECEPTORS

2.1. MOLECULARBIOLOGYOF GABAA RECEPTOR-CHANNELCOMPLEX Recent investigations into the molecular biology and biochemistry of the GABAA receptor have produced substantial advances in our understanding of its structure. The GABAA receptor is a heteroligomeric complex which comprises binding sites for GABA and for its allosteric modulators together with an integral ion channel. The GABAA receptor belongs to the super-family of ligand-gated ion channels, which includes the nicotinic acetylcholine receptor and the glycine receptor (Barnard et al., 1987). Solubilization of GABAA receptor complexes from mammalian brain membranes, followed by purification by benzodiazepine affinity chromatography, has yielded homogeneously purified receptors (for a review see Stephenson, 1988, and Stephenson and Dolphin, 1989). Partial sequencing of these pure protein preparations allowed the construction of oligonucleotide probes and ultimately the cloning and sequencing of the constituent subunits of the GABAA complex (Schofield et al., 1987). The native GABAA receptor is probably a pentamer (Stephenson, 1988) formed by integral membrane proteins belonging to at least four classes, ~, fl, 7 and 6. Indeed, a recent study (Schofield, 1989) suggests that as many as 13 different constituent proteins, belonging to 5 distinct subunit classes, may exist. At least three subtypes of the ~ and fl subunits and two of the 7 subunits have been cloned so far (Levitan et al., 1988; Pritchett et al., 1989a). The sequences of these proteins are markedly homologous and share the more general properties of the nicotinic acetylcholine receptor super-family (Schofield et al., 1987). These include the presence of 4 hydrophobic regions, presumably spanning the membrane (M I-M4), and other strongly conserved features, such as a ]/-structural extracellular loop containing N-glycosylation sites. Unlike the nicotinic receptor, however, the GABAA subunits lack the amphipathic MA helix between M3 and M4. Subunit classes differ in their molecular weights (48-53 kDa for ~, 55-57 kDa for fl and 48-50 kDa for 7; note that

GABA R~CI~I'TORMECHANISMS the value given for the ? subunit refers to the mature polypeptide and does not include possible carbohydrate moieties; Stephenson and Dolphin, 1989) and biochemistry: the intracellular M3-M4 region, which is poorly conserved, bears phosphorylation sites in the /31 and 72 subtypes (a serine and a tyrosine, respectively), but not in the ct subunits (Schofield et al., 1987; Pritcbett et al., 1989a). The /3 subunit phosphorylation site can be phosphorylated in vitro by cAMP-dependent protein kinase A (Kirkness et al., 1989). Differences between subtypes belonging to the ct class appear to be in the intracellular M3-M4 region and in the extreme N-terminal domain (Levitan et al., 1988). Photoaffinity labelling with muscimol is selective for the/3 subunits (Stephenson, 1988), whereas flunitrazepam photolabels only subunits which migrate electrophoretically in the ~t range (hence ct and/or 7; Pritchett et al., 1989a). The precise stoichiometry of the GABA A receptor is still unknown. The molecular weight of the purified receptor (about 240-250kDa) vs the molecular weights of the single subunits suggests a pentamer structure, while electrophoretic studies indicate that ~t and /3 subunits are present in equal amounts (Stephenson, 1988). Nevertheless, the likelihood that ? subunits co-migrate electrophoretically with ct subunits makes it difficult to establish the subunit composition of the native receptors. Furthermore, the levels of expression for the different subunits, and indeed for the different subunit variants, may be dependent on developmental stage and on the CNS area under consideration. For instance, the levels of m R N A coding for the ~ 3 subunit have been shown by Northern blot techniques to decline with development in bovine brain (Levitan et aL, 1988). In the adult CNS the m R N A for ct I appears to be the most abundant of the m R N A coding for the ~t variants. In situ hybridization studies indicate that ~t and 13 subunit RNAs are present in areas and cell subsets known to contain high densities of GABA A receptors, like the cerebral cortex, the hippocampus and the cerebellum of the rat (S6quier et al., 1988). The relative intensity of hybridization with ~t vs/3 probes, however, changes from one area to the other, the ct signal being much stronger than the/3 in the substantia nigra, with the opposite situation in the bed nucleus of the stria terminalis. Although regional variations in the relative stability or in the transcription and turnover rates of the ct and /3 RNAs may account for these observations, it is possible that these results reflect real differences in subunit composition (S6quier et al., 1988). Interestingly, one of the ? variants, 7 l, is expressed in glia (Pritchett et al., 1989a). Finally, the in situ hybridization pattern for the 72 subunit RNA does not appear to match entirely those for the ct and /3 subunits: in fact, ~2 expression is not prominent in the rat superior colliculus, which strongly reacts with ~t and /3 probes (Pritchett et al., 1989a; S6quier et al., 1988). RNA for the 6 subunit is present in a set of interneurones completely different from those expressing ~ 2 mRNA (Schofield, 1989). The distribution of the ~5 subunit appears to approach that of high affinity [3H] muscimol binding sites (Shivers et aL, 1989). These regional and developmental differences in subunit composition have important functional im-

37

plications for the pharmacology of GABAA receptors. Indeed, only the presence of the 72 subunit together with both the ct and fl subunits confers sensitivity to benzodiazepines and related agents to the assembled receptor complex (Pritchett et al., 1989a). A recent study (M6hler et al., 1989) has shown that GABA-gated currents mediated by ~t +/3 oligomers expressed in Xenopus oocytes are enhanced by diazepam despite the absence of the ? 2 subunit. This benzodiazepine response has, however, a peculiar pharmacology, because of the high concentrations of diazepam (# M range) required and because the potentiation is elicited by many benzodiazepine ligands, regardless of their properties of inverse agonists, antagonists or agonists in other systems. Verdoorn et al. (1989) have reported that expression of the ?2 subunit in addition to the ~1 or to the ~t1 + fl 1 in human embryonic kidney cells profoundly affects the desensitization of whole cell GABA currents and the single channel main conductance. On the other hand, substitution of one ~ variant for the other in ct + fl receptors expressed in oocytes does not change the electrophysiological properties of the receptor complex, but markedly affects the sensitivity of the receptor to GABA agonists. The EDs0 value (agonist concentration required to produce halfmaximal response) for whole-cell GABA-evoked peak currents was 1.3, 12 and 42/~M for receptors expressing the ~t2, a 1 or ~ 3 subunit respectively. This variation could be due to a change in receptor affinity, efficiency in recognition site--channel coupling or desensitization kinetics (Levitan et al., 1988). Furthermore, expression of the ~ 1 (rather than the ~t2 or ct3 variants) in combination with the/31 and ~2 subunits confers to the benzodiazepine site of the receptor complex the binding properties of a type I rather than a type II GABA-benzodiazepine receptor as shown by the greater sensitivity to the triazolopyridazine CL 218872 (see Section 2.2.2; Pritchett et al., 1989b). While all the available evidence indicates that the native GABAA receptor is heteromeric (i.e. constituted of different subunits), expression of pure ct,/3, 7 or t5 subunits has been found to yield homomeric receptor complexes that are functional and display normal channel properties and pharmacology, except for benzodiazepine sensitivity (Blair et al., 1988; Shivers et al., 1989). These homomeric receptors, however, are expressed or assembled less efficiently than heteromeric complexes both in Xenopus oocytes and in human embryonic kidney cell cultures (Blair et aL, 1988; Pritchett et al., 1988). Finally, part of the GABA A receptor heterogeneity could be due to differences in post-translational processing of the same subunit, or variant thereof, by glycosylation and by phosphorylation (Moffett et al., 1989; see below for a possible role of these processes in receptor regulation). 2.2. THE GABA A RECEPTORBINDINGSITES 2.2.1. GABAA recognition sites The precise location of the recognition site for GABA within the receptor complex has not been precisely established yet. Indeed, the finding that homomeric receptors are functional (Blair et al.,

38

L. SIVILOTTIand A. NISTRI

1988) indicates that each subunit type bears a site that can function as the recognition site. Where this recognition site may be located in a native beteromeric receptor is, of course, a different issue. Photoaffinity labelling with [3H] muscimol is known to label the fl subunit selectively (Stephenson, 1988). While this binding activity displays some of the characteristics expected of the recognition site (e.g. its bicuculline sensitivity), estimates of its dissociation constant are not reliable because of the irreversibility of photolabelling. Furthermore, only 7.5% of total receptor binding activity is labelled in this process (Casalotti et al., 1986). The general properties of the recognition site for GABA have been extensively investigated with radioligand binding techniques (for a review see Sharif, 1985). Early studies have demonstrated the uneven distribution of binding sites for [3H] G A B A in the CNS (Zukin et al., 1974) after blocking GABA uptake mechanisms with Na÷-free media (Enna and Maggi, 1979). Under these conditions, a single population of sites can be observed in crude membranes from rat brain, with a K0 value (dissociation constant) for [3H] GABA of 0.1-0.4pM at 4°C (Zukin et al., 1974; Enna and Snyder, 1975). Given that in CNS tissue GABA can reach concentrations of up to 1.91 pmol/g wet weight (Perry and Hansen, 1978), it is important to remove as much as possible of the endogenous GABA from the membrane preparations to be tested. This can be accomplished by several protocols, which include extensive washing, freezethawing, detergent treatments, dialysis and osmotic shock (or combinations thereof): these necessarily cause varying degrees of disruption in brain membranes, making it difficult to compare results obtained with different membrane preparations. The picture emerging from studies employing these techniques is altogether more complex than could be foreseen on the basis of the early results with fresh membranes. At least two classes of binding sites for [3H] GABA can be demonstrated at 0°C, with ranges o f K d values for GABA of 4-28 nu and 27-470 nu for the high- and low-affinity sites, respectively (Sharif, 1985; Olsen et aL, 1984). Gardner et al. (1981) have shown that the persistence of comparatively low levels of endogenous GABA (20-30/~U) in the membrane preparation can prevent detection of the high affinity binding sites. This observation could explain the early reports of a single population of binding sites. The GABAA agonist muscimol can also be used as radiolabelled ligand in receptor binding assays (Snodgrass, 1983) with the advantages over GABA of a low affinity for uptake sites (50% of GABA uptake inhibition is observed at the relatively high muscimol concentration of 240/IM; Krogsgaard-Larsen et al., 1984) and for GABA Breceptors (0.3% of the potency of GABA in displacing [3H] baclofen binding; Bowery, 1982). Like GABA, [3H] muscimol binds to two populations of sites with Kj of 21 and 365 nu in fresh, osmotically-shocked, well washed brain membranes (Yang and Olsen, 1987a). In solubilized purified receptor preparations one or more populations of sites can be demonstrated, depending on the nature of detergent used in the solubilization (Sigel and Barnard, 1984). Thus, receptors solubilized with Tri-

ton X-100 displayed one population of binding sites for [3H] muscimol (Kd = 33 riM), whereas receptors solubilized with the zwitterionic detergent CHAPS had a complex binding behaviour. Im et al. (1989) have recently described the immobilization of purified GABAA receptors onto concanavalin A-sepharose or polylysine agarose beads: this preparation can then be used for binding assays and shows one population of muscimol binding sites, whose K~ is 2.96 or 6.69 riM, depending on the detergent used, polidocanol (similar to Triton) or CHAPS, respectively. The same authors reported single-site muscimol binding in freeze-thawed, dialyzed membranes (Kd 9.14nM; Im et al., 1989). The properties of [3H] muscimol binding may be temperature-dependent: assays carried out on fresh membranes at 22°C reveal binding sites with a lower affinity than at 0°C (Kd 0.2-0.3 pM; Yang and Olsen, 1987a). Kinetic analysis of association and dissociation rates at 22-23°C with fast filtration techniques has confirmed the heterogeneity of muscimol sites, which may indeed comprise two high affinity sites (Kd 16 and 82 riM) and one low affinity site (Kd about 0.5 # u ) in frozen-thawed membranes (Agey and Dunn, 1989). The kinetics of muscimol binding are, however, quite complex and there are contrasting reports on the effects of causing the dissociation of receptor-ligand complexes by addition of excess cold ligand rather than by dilution (Yang and Olsen, 1987a; Agey and Dunn, 1989). It is at present very difficult to interpret these binding data in terms of actual heterogeneity of functional G A B A A receptors or receptor states. The existence of more than one class of binding sites leaves unanswered the question of their physiological relevance and raises several issues. For example, all binding studies give Kd values for agonists which are at least one order of magnitude smaller than EDs0 values observed in functional assays. The effects of detergent on muscimol binding to purified receptors (Sigel and Barnard, 1984), which should be free from contamination by endogenous substances, suggest that changes in the lipid environment of the receptor are sufficient to cause the appearance of more than one binding site (Lloyd and Beaumont, 1980; see also Im et aL, 1989). Note that the composition of the buffer used in the binding assay may affect the results of the assay (see, for an example, Tunnicliff and Smith, 1981). Different protocols for membrane preparation may result in differences in the levels of intracellular metabolites in the pinched-off vesicles that constitute particulate membranes. Given that GABA receptor properties can be profoundly affected by changes in their level of phosphorylation (see Section 5), it is not surprising that different membrane preparations vary in their binding behaviour. Richter and Curtis (1989) have indeed shown that addition of ATP to the incubation medium increases specific [3H] GABA binding. In addition, it is not inconceivable that, during the radioassay incubation, time membrane preparations may release trapped endogenous substances potentially active on the GABA receptor complex (Gardner et al., 1981) and that the release rates for such compounds depend upon temperature, ionic environment, etc. Another so far unresolved issue is whether the different sites are interconvertible, e.g. states of

GABA RECEPTORMECHANISMS

the same receptor. If such interconversion can take place under physiological conditions, differences in affinity may be linked to differences in the functional state of the receptor--channel complex (e.g. open, closed, desensitized; Olsen et aL, 1984). Conversely, the reported differences in G A B A EDs0 values due to variations in the subunit composition of the receptor (Levitan et al., 1988) point to a molecular basis for receptor heterogeneity which could explain some of the binding site heterogeneity. Finally, while most functional studies of the GABA receptor in the mammalian CNS suggest the existence of significant cooperativity (Nistri and Constanti, 1979), unity Hill slopes have been reported for the binding of agonists to the GABA recognition site (0.8-1.2, depending on the temperature; for muscimol, Yang and Olsen, 1987a). The reasons for such discrepancies are unclear, but it is possible that the membrane pretreatments, the non-physiological ionic environment and the protracted exposure to agonist substances which are necessary for binding studies may account for some of the quantitative differences. Biochemical studies of the receptor complex have so far failed to establish the precise number of agonist binding sites per receptor (Stephenson, 1988). Despite the limitations outlined above, the technique of ligand binding to isolated membranes has been useful in the screening and study of compounds that act on the GABA recognition site. GABAA agonists have been developed taking as initial models the chemical structure of GABA itself or of muscimol (for a review see Krogsgaard-Larsen et al., 1984). These lines of research have yielded a considerable number of active compounds, the most potent of which are the following (in brackets their IC50 in nM as inhibitors of GABA binding to rat brain membranes according to Krogsgaard-Larsen et al., 1984): dihydromuscimol (9), thiomuscimol (19), piperidin-4-sulphonic acid (P4S; 34), isoguvacine (37), trans-aminocrotonic acid (80), 3-aminopropanesulphonic acid (3APS; 80) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; 130). Although none of these compounds is more potent than muscimol (IC50 6 nM) in displaying GABA binding, some (notably P4S, isoguvacine and thiomuscimol) have an even lower affinity for GABA uptake mechanisms, while some (thiomuscimol) may penetrate the blood-brain barrier better. The most important antagonist of GABA acting on the GABAA recognition site is bicuculline, a phthalide-isoquinoline alkaloid found in several Fumariaceae (Henry, 1949). Curtis et aL (1970a) have shown that this compound depresses the inhibition evoked by iontophoretic GABA applications to cat Renshaw cells. The ICs0 value for bicuculline against GABA binding is 5/~ra (Zukin et al., 1974). Some of the major disadvantages of bicuculline, namely its low solubility and its instability at physiological pH (with a hydrolysis time course of a few minutes at 37°C; Olsen et aL, 1976) were overcome by the development of a quaternary analogue of bicuculline, the methiodide derivative (Johnston et al., 1972). Nevertheless, this compound also possesses some of the non-specific effects of the parent substance, including the ability to broaden the calciumdependent spike in mouse cultured spinal cord

39

neurones (Heyer et al., 1981). Indeed, bicuculline methiodide is more potent than the tertiary base in producing another unspecific effect, namely acetylcholinesterase inhibition (IC50 22 vs 43 #M; Olsen et al., 1976). The only other GABAA antagonist acting on the recognition site is pitrazepin (G/ihwiler et al., 1984), which has been suggested to be a competitive GABAA antagonist in the rat hippocampus (Kemp et aL, 1986). Binding studies indicate that pitrazepin is about 10 times more potent than bicuculline in displacing bound [3H] muscimol, but higher concentrations of pitrazepin also displace bound flunitrazepam (G~hwiler et al., 1984). In addition, pitrazepin is not a selective antagonist of GABA vs glycine in iontophoretic studies of cat spinal neurones (Curtis and Gynther, 1986). The binding of GABA and GABA agonists to the recognition site can be affected by the presence of other pharmacological agents which act on distinct sites of the GABA receptor complex, notably barbiturates, steroids and benzodiazepines (for reviews see Olsen, 1982; Olsen et al., 1986; Simmonds and Turner, 1987). Barbiturates markedly increase the Bmax value (maximal number of binding sites) for GABA binding: this effect is stereospecific and strongly anion-dependent, e.g. can be demonstrated only in the presence of halide ions (Willow and Johnston, 1981; Whittle and Turner, 1982). Due to the close correlation between barbiturate hydrophobicity and their potency in enhancing GABA binding, it has been suggested that this effect is due to a physical action of barbiturates dissolved in the lipid phase of the cell membrane (Simmonds and Turner, 1987), rather than to a direct effect of barbiturates on the GABA receptor. A facilitation of GABA binding distinct from that caused by barbiturates is elicited by anaesthetic steroids, which increase the affinity of [3H] muscimol binding to mammalian CNS membranes (Harrison et al., 1987) perhaps by producing a perturbation in the lipid phase of the membrane, like barbiturates are proposed to do (Simmonds and Turner, 1987). As for benzodiazepine agonists, their enhancement of GABA binding is fairly small and limited to the low affinity component (Skerritt and Johnston, 1983). Other authors (Guidotti et al., 1978) have described a similar effect on high affinity sites only under special conditions of membrane preparation. It is interesting to note that not only pure antagonists at the benzodiazepine site, but also inverse agonists do not affect GABA binding (Simmonds and Turner, 1987). 2.2.2. Benzodiazepine sites Another important site present in the GABA receptor complex is the one for benzodiazepines. This appears to be situated on the y2 subunit of the receptor (Pritchett et al., 1989a). At present it remains unclear whether this site is physiologically activated by an endogenous ligand, although various substances have been suggested as candidates for this role (for reviews see Turner and Whittle, 1983; Mathers, 1987; Barbaccia et al., 1988). Stereospecific, saturable binding of [3HI diazepam to rat brain membrances was first reported by Squires

40


and Braestrup (1977) and by Mfhler and Okada (1977). The K~ for diazepam at this site is 2.6-3.6 riM; the potency of various benzodiazepines in displacing it correlates well with their muscle relaxant activity (Squires and Braestrup, 1977). Although the interaction of most benzodiazepines with the [3H] diazepam binding site follows the mass action behaviour expected for a single population of binding sites, a few compounds, notably the triazolopyridazine CL 218872 and the methyl, ethyl and propyl esters of fl-carboline-3-carboxylic acid displace bound [3H] diazepam with Hill slopes significantly less than unity in hippocampus (Sieghart, 1989). In addition, direct binding of [3H] CL 218872 yields curvilinear Scatchard plots (Yamamura et al., 1982). These results indicate the existence of two distinct benzodiazepine receptor subtypes (type I and type II; Klepner et al., 1979), although other interpretations (such as the existence of interconvertible conformational states) are equally plausible, in the light of the complex kinetic behaviour of the benzodiazepine receptor with any ligand (for a review see Martin et al., 1983). Fitting of a two-site model to the data suggests that the selectivity of CL 218872 is not very high, with an affinity for type I sites approximately 21-fold greater than that for type II sites at 0°C. Raising the temperature to 37°C increases the Hill slope from 0.66 to 0.82 and decreases the selectivity of CL 218872 to only a 7-fold preference for type I sites (Gee et al., 1983). The case for benzodiazepine receptor heterogeneity has been strengthened by the recent demonstration that cloned ctfl172 GABA receptors manifest type I or type II binding properties depending on the ct variant expressed (Pritchett et al., 1989b). GABA receptors containing the ct 1 variant resemble type I receptors in their affinity for fl-carboline-3-carboxylate methyl ester and CL 218872, whereas receptors expressing ct2 or ~3 subunits behave like type II receptors. The affinity ratio of CL 218872 for the ctl- vs the ct3-containing cloned receptors is affected by temperature changes in the same way as that for type I vs type II receptors in brain membranes. The predominance of ~ 1 activity in RNA from adult cerebellum and of ~3 in RNA from newborn brain tissue (Pritchett et al., 1989b) agrees with the levels of type I and type II receptors previously reported by binding studies on the same preparations (Young et al., 1981; Eichinger and Sieghart, 1986). Contrary to expectations, peripheral tissues were also found to contain high levels of binding sites with nanomolar affinity for tritiated benzodiazepines. These "peripheral" sites, however, display distinctive binding selectivity which differentiates them from the "central" sites (for a review see Verma and Snyder, 1989). In particular, the anticonvulsant benzodiazepine clonazepam, which is very potent in displacing [3H] diazepam from central sites (IC50 1.5-5nM; M6hler and Okada, 1977; Squires and Braestrup, ! 977), is very weak at peripheral sites (IC50 5.7/~i; Schoemaker et al., 1983). Conversely, another benzodiazepine derivative, Ro 5-4864 (4'chlorodiazepam, which possesses convulsant activity) and the isoquinoline carboxamide compound PK 11195 bind to the "peripheral"-type benzodiazepine recep-

tors with affinities at least two orders of magnitude greater than those for central sites (Squires and Braestrup, 1977; Schoemaker et al., 1983; Le Fur et al., 1983). These "peripheral" sites are present in a variety of tissues (including brain; Anholt et al., 1985) and appear to be associated not with the GABA A receptor complex but with the voltage-dependent anion channel of the outer mitochondrial membrane (Verma and Snyder, 1989). The picture is complicated by the recent description of a second binding site for Ro 5-4864, which may be located on the GABA A receptor itself, but is distinct from the "central" benzodiazepine site and may mediate the excitatory effect of Ro 5-4864 in a mammalian cell line transiently expressing ct lf1172 GABA receptors and in rat cultured neurones (Gee, 1987; Gee et al., 1988). The results of these binding studies have recently been confirmed by Puia et al. (1989), who have shown that Ro 5-4864 decreases whole-cell GABA currents in a manner insensitive to the benzodiazepine antagonist flumazenil (Ro 15-1788). The existence of a second "central" binding site with micromolar affinity for diazepam (Kd = 45/~M) was reported by Bowling and De Lorenzo (1982), who suggested that this site may mediate the inhibitory effect of benzodiazepines on calcium-calmodulin protein kinase and on voltage-sensitive calcium channels (Taft and De Lorenzo, 1983) and may be responsible for the benzodiazepine-induced protection from electroshock seizures. The binding of benzodiazepines and inverse agonists to the benzodiazepine recognition site can be modulated by a variety of compounds acting on any of the pharmacological sites of the GABA receptor complex. Firstly, GABA itself has been shown by binding studies to produce a 2-fold increase in the affinity of [3H] diazepam for its binding sites. This effect of GABA, also known as GABA-shift, is dependent upon the presence of C1 in the incubation medium and is antagonized by bicuculline (Tallman et al., 1978). This effect of GABA is shared by all GABAA agonists, some of which (3 APS, isoguvacine) behave as partial agonists (Braestrup et al., 1979; Williams and Risley, 1979). The ranking of GABA agonist activity in enhancing benzodiazepine binding and in displacing bound GABA (or muscimol) is very similar, but their potency in affecting the benzodiazepine site is considerably lower (GABA itself shows an ED~0 value of 1.1-1.6/~M). A positive GABA-shift can be demonstrated only when benzodiazepine agonists are used. The binding of the benzodiazepine antagonist flumazenil (Ro 151788) is not affected by GABA (M6hler and Richards, 1981), whereas that of inverse agonists like methyl-fl-carboline-3-carboxylate, shows a lower affinity (negative GABA shift) in the presence of GABA (Braestrup and Nielsen, 1981). [3H] Diazepam binding activity is also enhanced by up to about 2.5 times by barbiturates in concentrations similar to those that enhance GABA binding. The barbiturate rank activities can be correlated with their relative potency as general anaesthetics. The action of barbiturates is stereospecific, enhanced by the presence of halide ions and is competitively antagonized by picrotoxin (Leeb-Lundberg et al., 1980).

GABA RECEPTORMECHANISMS On the other hand, picrotoxin per se has no effect on diazepam binding (Tallman et al., 1988; Williams and Risley, 1979) unless the assay is performed in fairly restrictive conditions (either 0°C in the presence of C1- or 35°C in Cl--free medium): in this case high concentrations of picrotoxin (100/~M) produce a moderate increase (20%) in the Bmax value for flunitrazepam binding (Karobath et al., 1981). It should be noted that the presence of Cl- ions, and generally of anions which permeate the GABA-gated channel, not only affects the action of modulators of benzodiazepine binding, but enhances benzodiazepine binding per se, with bell-shaped dose-response relationships (Costa et al., 1979). Both actions of anions are strongly temperature-dependent, Benzodiazepine binding was found to be stimulated by the anthelmintic lactone avermectin B~a at nanomolar concentrations; the effect of avermectin BI a was enhanced in the presence of Cl- or picrotoxin and reduced by GABA A agonists (Drexler and Sieghart, 1984). Finally, anaesthetic steroids enhance flunitrazepam binding affinity (Harrison et al., 1987). 2.2.3. Picrotoxin/cage convulsant sites Another pharmacologically important site on the GABA receptor complex is the site that mediates the antagonist effects of picrotoxin. This substance is obtained from the seeds o f A n a m i r t a cocculus (Menispermaceae) and is a l : l molecular compound of two dilactones, i.e. the active principle picrotoxinin, and the inactive picrotin. A derivative of picrotoxinin, [3H]ct-dihydropicrotoxinin (DHP), can be used as a ligand for this site in binding studies, but its low affinity (1-2 #M) causes a poor ratio of specific/nonspecific binding (Olsen, 1982). The cage convulsant [35S] t-butylbicyclophosphorothionate (TBPS) has a considerably higher binding affinity than DHP for picrotoxin sites (25-50 riM; Squires et al., 1983; Ramanjaneyulu and Ticku, 1984). TBPS and other cage convulsants like pentylenetetrazole, bemegride and bicyclophosphates competitively inhibit the binding of [3HI DHP (Ticku and Olsen, 1979) and, conversely, picrotoxin is a competitive inhibitor of TBPS binding (Lawrence and Casida, 1983). Observed differences in the Bma~ values obtained using DHP or TBPS as ligands can be interpreted as due to an incomplete overlap between the sites labelled by the two probes, or, more economically, as the result of the wide margins of error caused by the low specific binding of DHP (10-15% of total binding; Ticku and Maksay, 1983). The binding of both DHP and TBPS is inhibited by barbiturates, including convulsant derivatives like dimethyl-butyl-barbituric acid (Ticku and Olsen, 1978; Squires et al., 1983). While the inhibition of DHP binding by barbiturates is competitive (Ticku and Olsen, 1978), TBPS binding inhibition by pentobarbitone is apparently not competitive (Ramanjaneyulu and Ticku, 1984). It must, however, be noted that the pentobarbitone concentration used in this study for fitting the Scatchard plot was rather high (200 #M). Functional assays have shown that at this concentation pentobarbitone adds to its GABAenhancing properties a direct GABA mimetic action

41

(Suzdak et al., 1986). Since GABA A agonists are known to affect TBPS binding (Squires et al., 1983), it is possible that 200 #M pentobarbitone may share this action. Conversely, the study which established the competitive nature of the pentobarbitone inhibition of DHP binding was carried out with a lower pentobarbitone concentration (50#M; Ticku and Olsen, 1978). Recent work by Abel et al. (1989) has shown that the magnitude and the direction of the interaction between TBPS binding and a variety of compounds acting on the GABA receptor complex (including GABA, barbiturates, benzodiazepines, fl-carbolines) are strongly dependent upon the assay protocol: in particular, the ionic medium and equilibrium or non-equilibrium conditions. At equilibrium and in the presence of Cl- ions, only barbiturates and GABA affect TBPS binding, producing a dose-dependent inhibition (Abel et al., 1989). Halide and other anions have been shown to increase the affinity of TBPS for its binding sites; the potency of anions in evoking this effect appears to be correlated with their permeability through GABA-gated channels (Havoundjian et al., 1986). Finally, Harrison et al. 0987) and Huidobro-Toro et al. (1987) have reported that TBPS binding (to crude synaptosomal membranes and to mouse brain microsacs, respectively) is dose-dependently inhibited by anaesthetic steroids. In the study carried out by Harrison et al., steroids were found to affect both the Kd and the Bmax of TBPS binding and, at concentrations subthreshold for a direct effect, to enhance the stimulatory effect of muscimol on TBPS binding. Nevertheless, it appears that the choice of membrane preparation determines the nature of the observed steroid response, which can consist of a pure depression throughout the dose range (in fresh membranes) or an enhancement at low doses and a depression at high doses (in frozen-thawed membranes; Turner et al., 1989). The effect of steroids is additive to that of pentobarbitone, but synergistic in the case of alphaxalone (Turner et al., 1989).

2.3. A FUNCTIONALASSAYOF GABA A RECEPTOR ACTIVITY: 36C1- FLUX MEASUREMENTS One major limitation in binding assays is that the parameter measured is simply the amount of ligand bound to a receptive site: no information is available as to whether the ligand site interaction can produce functional changes in cell properties. While electrophysiological methods can provide a direct measure of receptor function in terms of changes in receptorgated ionic permeability, an alternative is to monitor with radiochemical tracer techniques the ionic flux induced or blocked by receptor activation. In the case of GABA receptors experimental methods rely on the fl-emitter 36C1-. Various types of brain particulate material, such as cortical "microsacs" (Harris and Allan, 1985) or filtered synaptoneurosomes (Schwartz et al., 1984), cultured neurones (Thampy and Barnes, 1984; Lehoullier and Ticku, 1989) and brain slices (Wong et al., 1984; Yang and Olsen, 1987b) have been studied. For brain slices the technique involves their incubation in 36C1- buffer and subsequently the

42


measurement of 36C1- effiux in the presence or absence of GABAergic drugs. For cultured neurones and subcellular preparations 3~C1- uptake is generally assayed (see however Schwartz et al., 1984) by incubating the preparation in 36C1- buffer containing the GABAergic drug to be tested and stopping uptake at a fixed time by addition of ice-cold medium and fast filtration. In principle, since CI- flux is the first event following GABAA receptor activation, estimating this ionic flow should provide a first-order response and should therefore be extremely useful in quantitative pharmacological studies of the GABA A receptor. Unfortunately, the signal measured by these assays (be it 36C1- uptake or 36C1- efflux), does not allow such developments for several reasons (Yang and Olsen, 1987b). Firstly, in many neurones the direction of the GABA-evoked CI- flow differs between cell body and dendrites, because of local differences in the CIequilibrium potential (Alger and Nicoll, 1982a; Barker and Ransom, 1978), making the observed responses composite. Furthermore, tissue preparations are normally heterogeneous, and include an unspecified proportion of glial cells (but see Thampy and Barnes, 1984), some of which may carry functional GABAA receptors and provide effective buffering of extracellular C1- (Bormann and Kettenmann, 1988; MacVicar et al., 1989). In some studies (Thampy and Barnes, 1984) the signal-to-noise ratio, namely the 36CI- uptake induced by G A B A over and above basal influx, is very poor in the presence of near physiological potassium concentrations (5.4 mM) and the experiments have therefore to be carried out in the presence of very high (40 mu) K + levels. In subcellular preparations, the problem of the heterogeneous origin of the structures involved in the CIfluxes is still present; in addition, it is likely that in these structures, be they microsacs, synaptosomes or synaptoneurosomes, the maintenance of physiological CI- gradients and the full functionality of the GABA A receptor is far from complete. Unspecified levels of endogenous G A B A may also be present in the "extracellular" compartment (Dunn et al., 1989; Schwartz et al., 1984) and GABA or other endogenous substances may leak from the vesicles during the experiment. On the other hand, slice experiments have the disadvantage of presenting restricted diffusion to substances and hence of greater uncertainty on the effective drug concentration in the receptor compartment. Furthermore, even in a recent study the efflux had to be measured at 1 min intervals, making it difficult to assess the true "instantaneous" rate of efflux without influences from receptor desensitization (Yang and Olsen, 1987a,b). Agonist exposure times for the other techniques can be very much shorter, and range from 3 (Harris and Allan, 1985) to 20 s (Lehoullier and Ticku, 1989). Particulate membrane preparations lend themselves to rapid mixing experiments, such as the ones described with the quench-flow technique by Cash and Subbarao (1987a,b): the fast reaction times for the start of agonist-induced ionic flow and for its termination by mixing with bicuculline methiodide (at a final concentration of 1.5 mM) have allowed these authors to study the kinetics of GABA-evoked 36C1- uptake and of GABA receptor desensitization.

A further improvement in the time resolution of anion flow assays has come by the recent development (Dunn et al., 1989) of a fluorescence quenching method using solubilized GABA receptors reconstituted into phospholipid vesicles. The washed vesicles are loaded with an anion-sensitive fluorophore, 6methoxy-N-(3-sulphopropyl)quinolinium, the extravesicular excess of which is removed by gel filtration. The quenching of the trapped fluorescent dye by CIor I - can then be followed by stopped-flow spectroscopic techniques. C1- flux assays have been used to investigate all aspects of the pharmacology of the G A B A A receptor. The potencies of agonists in these studies are within the range of values obtained with electrophysiological methods and vary by about one order of magnitude between one preparation and the other. The EDs0 values for GABA range between 1.3/tM in chick cerebral neurones in culture (Tehrani et al., 1986) and 13/~u in brain microsacs (Harris and Allan, 1985). Other authors have used muscimol as the GABA A agonist of choice, reporting EDs0 values of 0.2-1 #M (for reconstituted vesicles: Dunn et al., 1989), 2/~M (for mouse spinal cord neurones in culture: Lehoullier and Ticku, 1989), 3/aM (for mouse cortical slices: Yang and Olsen, 1987b) and 8 #M (for chick neurones in culture: Thampy and Barnes, 1984). Thampy and Barnes (1984) have also tested GABA in their preparation, obtaining an EDs0 value of 6/~M (compared with the ED~0 value for G A B A and muscimol reported by Harris and Allan in 1985 of 13 and 5/~M, respectively). Thampy and Barnes (1984) have also tested 3-aminopropanesulphonic acid (3APS), which was equiactive with GABA at 50/~M, and taurine, glycine and 2,4-diaminobutyrate, which were all inactive at 50/~M concentration. Hill slopes near unity were observed for GABA or muscimol in most of these assays (for a review see the discussion in Yang and Olsen, 1987b), with the exception of Yang and Olsen's study on mouse cortical slices, which found a slope significantly shallower than unity for muscimol. It must be borne in mind that the presence and extent of desensitization in these experiments is likely to affect the Hill slope value. The same authors described a decline in the response to muscimol with prolonged exposure to the drug: this was already apparent in the second 1-min period of collection. GABA-stimulated 36C1 fluxes were found to be bicuculline- and picrotoxin-sensitive by the majority of the investigators (Wong et al., 1984; Thampy and Barnes, 1984; Harris and Allan, 1985; Dunn et al., 1989; Lehoullier and Ticku, 1989). In filtered synaptoneurosomes (Schwartz et al., 1984), but not in brain microsacs (Harris and Allan, 1985), picrotoxin has been found to decrease basal 36C1 flux: these data probably indicate the presence of suprathreshold levels of endogenous GABA. The IC50 value for bicuculline was found to be 3.5gM (Thampy and Barnes, 1984) or 4.5 pM (Lehoullier and Ticku, 1989) in neuronal cultures, while corresponding values for picrotoxin (Thampy and Barnes, 1984) and for picrotoxinin (Lehoullier and Ticku, 1989) were 1 and 25/~M respectively. The nature of drug antagonism was investigated by Thampy and Barnes (1984), who found it non-competitive for bicuculline and uncompetitive for picrotoxin. In the same preparation,

GABA RECEPTOR MECHANISMS

TBPS non-competitively antagonized the GABAevoked 36C1- inflow (IC50=0.30#M) much more potently than the basal 36C1- inflow (IC50 value = ll.4/aM; Tehrani et aL, 1986). In most of these studies (with the exception of Dunn et al., 1989) barbiturates were found to be pharmacologically active. At low concentrations (10/aM; Harris and Allan, 1985) pentobarbitone enhanced the effect of GABA on CI- fluxes, whereas at higher concentrations (0.3mM; Harris and Allan, 1985; Yang and Olsen, 1987b) pentobarbitone induced an increase in CI- fluxes per se with an estimated EDs0 value of 0.2 mM (Suzdak et al., 1986). Yang and Olsen (1987b) have observed that 0.3 mM pentobarbitone not only evoked 36C1- efflux from slices, but also shifted the muscimol dose-response curve to the left and upwards. Benzodiazepines are generally inactive on CIfluxes per se, but readily enhance the effects of GABA or muscimol (Obata and Yamamura, 1986; Yang and Olsen, 1987b; Mehta and Ticku, 1989). Such an enhancement can be observed only with submaximal test concentrations of GABA or muscimol: in mouse cultured spinal neurones the most potent compound is flunitrazepam (EDs0 value = 133 nM), followed by clonazepam (175 riM), diazepam (450 riM) and flurazepam (1.5/aM; Mehta and Ticku, 1989). The GABA response is potentiated by benzodiazepines by up to 100%. A smaller enhancement of GABA responses (up to 50%) plus higher benzodiazepine potencies (ED m = 23 nM for flunitrazepam) were observed by Obata and Yamamura (1986) using brain microsacs in the presence of diazepam, flunitrazepam or clonazepam. Flumazenil (Ro 15-1788) reduced the effect of GABA at concentrations lower than 1/aM, but curiously shared the enhancing action of benzodiazepine agonists at higher doses (EDs0 3.9#M; Mehta and Ticku, 1989). Both Obata and Yamamura (1986) and Mehta and Ticku (1989) found that flumazenil antagonized the effects of benzodiazepine agonists and inverse agonists on the GABA response, although its blocking activity differed considerably in these studies. Inverse benzodiazepine agonists, including methyl 6,7-dimethoxy-4-ethyl-fl-carboline-3carboxylate (DMCM), are reported to have no effect on CI- fluxes in cortical slices (Yang and Olsen, 1987b), whereas they reduce the response to GABA by up to about 50% in brain microsacs (IC50 for DMCM 3 nM; Obata and Yamamura, 1986) and in mouse spinal neurones in culture (Mehta and Ticku, 1989). The effects of GABAergic agonists on 36C1fluxes are also enhanced by a variety of anaesthetic agents, including alphaxalone and related steroids (Turner et al., 1989), diethylether and chloroform (Huidobro-Toro et al., 1987). Alphaxalone (10/aM) was found to potentiate near-maximal responses to muscimol in hippocampal slices: at this concentration the steroid had little or no effect on C1- fluxes p e r se (Turner et al., 1989). Majevska et al. (1986) reported that metabolites of progesterone and deoxycorticosterone evoked a picrotoxin-sensitive increase in basal 36C1- uptake into brain vesicles at concentrations as low as 300 nM (for 3-c¢-hydroxy-5~t-dihydroprogesterone). Chloroform and diethylether enhanced only the GABA-dependent 36C1- uptake into brain microsacs (Huidobro-Toro et al., 1987): the dose-response

43

curve for this effect of diethylether appears to be bell-shaped with a peak at 100 mM. 3. E L E C T R O P H Y S I O L O G Y OF GABA A R E C E P T O R MECHANISMS

As indicated earlier, a direct approach to the study of GABAA receptor-mediated responses is provided by electrophysiological experimental techniques. Such methods have produced considerable insight into the ionic permeability of the GABA-activated anion channel and its conductance. 3.1. IONIC SELECTIVITY OF G A B A A RECEPTORACTIVATED CHANNELS

Establishing the ion permeability of chemicallygated channels provides information on the molecular structure of these channels and on their role in synaptic potentials. In particular, such studies can supply an estimate of the size of the open channel and can reveal the presence of binding sites for permeant ions on the inner pore walls. It is clear that C1 is the ion responsible for the effects mediated by GABAA receptor activation (Bormann, 1988). The traditional approach used to investigate the CI- channel properties is the one pioneered by Eccles and co-workers on cat spinal motoneurones in vivo (Coombs et al., 1955): the reversal potential of the synaptic potential to be studied (in the case of Coombs et al., the glycine-mediated Cl--dependent IPSP of spinal motoneurones) is measured before and after intracellular injection of a chosen anion. If the channels opened during the IPSP are permeable to the injected anion, the IPSP reversal potential (Ewsp) should be shifted towards a new equilibrium potential by this anion, by a factor dependent on the permeability of the anion relative to that of CI-. This method has also been applied to GABAergic IPSPs in the neocortex (Kelly et al., 1969) and in the hippocampus (Eccles et al., 1977). The technical difficulties of such an approach, particularly with in vivo preparations, are obvious. Furthermore, the reliability of this test in determining whether an anion is permeant or not through the CI- channel depends on the fulfilment of at least two assumptions. Firstly, the synaptic signal must be mediated by a homogeneous population of receptorgated channels. Therefore, any contamination by EPSPs or by GABAa-mediated IPSPs would invalidate the results. The second and more important assumption is that any change in EiPsP must result only from the introduction of new terms relative to the injected anion A - (without altering pre-existing values) into the Goldman-Hodgkin-Katz equation (i) which describes the general behaviour of an ionmediated synaptic potential: RT

EiPsp = - - +

F

In

[Cl-]o + PA/PcI[A-]o [Cl-]i + PA/PcI[A-]i

(1)

where PA and Po are the permeabilities to A and C1- and the other symbols have their customary meaning. However, as shown by Eccles and coworkers (Allen et al., 1977), the injected anion A may affect the intracellular C1- activity [CI-]~, and

44

L. S1VILOTTIand A. NISTRI

thus the transmembrane distribution of C1 . Even anions completely impermeant through the synaptic channel might produce a depolarizing shift in the reversal potential of the IPSP if they increase [C1-]~. Such secondary effects of the injected anion could conceivably be due to interference with one of the C1 pumps which are known to maintain the Cl- distribution in central neurones (Allen et al., 1977) or with K÷/Cl exchange mechanisms (Thompson et al., 1988) and cannot be excluded, unless independent measurements of [Cl-]i are obtained. Finally, recent work on crayfish muscle fibre (Kaila and Voipio, 1987; Kaila et al., 1989) and on mouse spinal neurones in culture (Bormann et al., 1987) has shown that GABA-activated channels are also permeable to bicarbonate ions (HCO3). In physiological conditions the reversal potential for GABA-evoked currents deviates significantly from the C1- equilibrium potential towards the HCO~- equilibrium potential (Kaila and Voipio, 1987; Kaila et al., 1989). In its turn, HCO3 is not passively distributed, but its intracellular level is determined by the pH regulatory mechanisms of the cell (Thomas, 1984). Any interference of the injected anion with the acid-base balance of the cell would result in a shift in the H C O f equilibrium potential and consequently in E~psp. A novel approach to the issue, which potentially overcomes the problems detailed above, is that of using patch-clamp techniques in order to manipulate the intracellular ionic environment. In principle, use of this method with excised patches offers full control over the composition of the intra- and extracellular media. This technique has been used in mouse spinal neurones in culture with the excised patch as well as the whole cell-clamp configuration (Bormann et al., 1987). In the latter mode the intracellular medium is dialyzed by a wide-bore patch pipette. The obvious advantage of this method over conventional ion injection via microelectrodes lies in the rate of ion exchange, which allows manipulations of ionic concentration over a wider range and in a much shorter time. It is also likely that any secondary changes in CI- or HCO3 concentrations would be buffered by the pipette solution. Indeed, in their calculations of intracellular ion concentrations, Bormann et al. assume the total replacement of the intracellular phase by the pipette solution. Being able to estimate the intra- and extracellular concentrations of permeant anions allows the measurement of the permeability of each anion relative to CI and to establish anion permeability sequences. The whole-cell patch-clamp data (Bormann et al., 1987) indicate that the GABA-gated channel is permeant to organic anions up to the size of propionate, including formate and acetate: all three have a permeability lower than that of Ct-, respectively 1.7, 50 and 8% of Pc~. The best fit for the relation of the relative permeability to the Stokes diameter (Dwyer et al., 1980) is given by a pore diameter value of 0.56nm, not dissimilar from the estimate of 0.5-0.8 nm obtained by Kelly et al. (1969) for neocortical neurones. The maximum size of organic ions found to be permeant through the CI- channel in the patch-clamp experiments is intermediate between that reported by Kelly et al. (1969) and by Eccles et al. (1977). In these studies, the largest permeant

organic ions were, respectively, glutamate and formate. It must be noted that acetate ions have an anomalous effect on spinal motoneurones, namely they can reduce or reverse the IPSP when diffusing into the cell, but not when injected by iontophoresis (Eccles et al., 1977). Substantial agreement was found between the three studies on the permeability of inorganic anions; the sequence (in order of decreasing permeability according to Bormann et al., 1987) is: SCN- > I - > N O 3 > B r - >NO~- > C l - > H C O £ > F - > HEPO 4 ; F - , which is 50 times less permeant than Cl- in the patch-clamp study, has slight permeability according to Kelly et al. (1969) or is impermeant according to Eccles et al. (1977). BrO3 and methylsulphate were found to be impermeant in both studies in vivo. The permeability sequence for inorganic anions found by Bormann and co-workers is the same as that predicted by the lyotropic series (Diamond and Wright, 1969). This indicates that the interaction of the ions with binding site(s) on the channel wall is weak, being better explained by their dehydration energy than their binding energy. Patch-clamping has also provided an estimate of the K ÷ permeability of GABA-activated Cl- channels: this appears to be very low, with an upper limit of 5% of the CI permeability. This parameter could not be reliably calculated in the past because the standard method, e.g. measuring the reversal potential for GABA in the presence of different extracellular K + concentrations, leads to secondary changes in intracellular Cl levels (Barker and Ransom, 1978; Alger, 1985). 3.2. CONDUCTANCEOF THE G A B A A RECEPTORGATED ION CHANNEL The first estimates for the conductance of the GABA-activated channel were provided by fluctuation analysis of GABA responses recorded with two electrode voltage-clamp from mouse spinal and sensory neurones in culture (Barker et al., 1982). The average single channel conductance (~) presented considerable variability, particularly in spinal cord neurones with a range from 3 to 25 pS (average 16 pS). The same technique used on rat hippocampal neurones in culture yielded values of 19.8 and 18.7 pS for the elementary conductance evoked by G A B A and muscimol, respectively (Segal and Barker, 1984a). Slightly smaller estimates (16 and 12pS, respectively) were obtained by Cull-Candy and Ogden (1985) and by Cull-Candy and Usowicz (1989) for rat cerebellar neurones in culture. In recent years, the technique of single-channel recording from membrane patches has been employed to study GABA-operated channels in a variety of preparations. In most studies the presence of multiple conductance states has been described (see Table l) with main levels in the region of 20 and 27-30 pS (Fig. 1). The reported differences can probably be accounted for by different animal species, tissue types and stage of development plus variations in the experimental protocol, particularly in the ionic environment of the patch. It should also be noted that, since some of the subconductance levels are comparatively rare, any estimate of their value is liable to error. Results obtained with the cell-attached


45

TABLE 1. VALUESOF MULTIPLECONDUCTANCESTATESEVOKEDBY GABA References

Tissue

Preparation

Single channel conductance

Bormann and Clapham, 1985

Bovine adrenal chromaffin cells in culture

Excised outside-out patches

45, 31, 18, 10

Gray and Johnston, 1985

Guinea-pig partly dissociated hippocampal neurones

Cell-attached and excised patches (mainly inside out)

23 (at - 4 0 mV)

Bormann et al., 1987

Mouse spinal neurones in culture

Cell-attached

28, 17, 10

Outside-out excised patches

44, 30, 19, 12

Bormann and Kettenmann, 1988

Rat astrocytes in culture

Outside-out excised patches

43, 29, 21, 12

Weiss et al., 1988

Chick cerebral neurones in culture

Outside-out and inside-out excised patches

27-32, 20.8, 10-13, 6-8

Blair et al., 1988

Xenopus oocytes

Inside-out excised patches

40-42, 27-28, 18-19, 10

expressing pure ~t and fl subunits Llano et al., 1988

Organotypic cerebellar cultures

Outside-out patches

28-30, 19-20

Macdonald et aL, 1989a

Mouse spinal neurones in culture

Outside-out patches

27.2, 15.9

Verdoorn et al., 1989

Human kidney cells expressing combinations of GABAA receptor subunits

Cell-attached and outside-out patches

~qfit 19.9 ~t~72 34.5 ~qfl~'2 36.4

Data are single-channel conductance (y) values expressed in pS. The most frequently occurring state in each preparation is in italics. configuration are also affected by the inaccuracies in determining the intracellular CI- concentration and the resting membrane potential (Bormann et aL, 1987; Bormann, 1988). A problem of patch-clamp techniques is that, with the exception of the cell-attached configuration, in which the membrane is not perforated, the intracellular compartment is dialyzed via the patch pipette. This can result in the loss of intracellular metabolites, which may be important for normal channel function. The degree of the loss depends on the configuration chosen and, for the whole-cell clamp, on the cell size (Ogden and Stanfield, 1987). Bormann et al. (1987) have also determined singlechannel conductances in patches exposed to CI- on the extracellular side and equimolar concentrations of other halide ions (or S C N - ) on the other. The conductance sequence found is C I - > B r - > I - > S C N - > F - , nearly the reverse of the permeability sequence, This indicates the presence of binding sites for the permeant ions in the channel, which was corroborated in the same study by the observation that the single-channel current-voltage relation deviates from constant field behaviour. In experiments carried out in excised patches with C I - / S C N - mixtures on both faces of the membrane, the behaviour of the single-channel conductance in response to changes in the mole fraction of S C N - is best predicted by a model that assumes the existence of two

binding sites for permeant ions on the channel walls. Studies carried out with fluctuation analysis agree in reporting that the elementary conductance of the GABA-activated CI- channel is not dependent upon the membrane potential over a fairly wide range of values ( - 1 0 0 - 2 0 mV, Barker et aL, 1982; - 7 0 + 10 mV, Segal and Barker, 1984a; - 110 + 50 mV, Cull-Candy and Usowicz, 1989). Similar results obtained with single-channel recording and excised patches held in symmetrical CI- media have been described for all the conductance levels observed (-100+60mV, Bormann et al., 1987; - 1 0 0 + 100mV, Bormann and Kettenmann, 1988) or for the main conductance state ( - 6 0 + 6 0 m V , Weiss, 1988; - 8 0 + 6 0 mV, Macdonald et al., 1989a). In a minority of studies, outward rectification of the single-channel conductance has been observed. In patches from partly dissociated neurones of the guinea pig hippocampus neurones, Gray and Johnston (1985) have found that only one conductance level for the channel could be resolved, with a 7 of about 20 pS at - 4 0 mV and nearly 40 pS at +40 mV in symmetrical CI- solutions. The reasons for this discrepancy are obscure, but could be linked to the experimental protocol, in particular the use of proteolytic enzymes to expose the cell body regions of the slices and the choice of preparation, namely adult nervous tissue. Outward rectification in the singlechannel conductance has also been described by Blair

46


.9E ,oo .0 O

6 z

k 0'S

14) •s Stepsize(DA)

2~o

CL

D I

.

.

.

.

.

.

.

.

[lpA

00

1

o-

z 0

0.5

1.0 1.5 Stepsize(pA)

2..O

rye,-

FIG. 1. Subconductance states of GABA and glycine channels recorded from cell-attached patches of mouse spinal neurones. Current traces represent plots of current recordings (outward current upwards) digitized at 0.1 ms/point. A: Glycine-activated channels with various substates in addition to the most frequent channel opening (1.39 pA, cf. at). Horizontal time calibrations: 20 ms for a t , 75 ms for a2 and 40 and 5 ms for a3(top and bottom, respectively).B: Distribution of glycine-activatedcurrents; note main peak at 1.4 pA. C: GABA-activated channels with various substates. Horizontal time calibration: 75 ms in c~ and c.,, 38 ms in ca (top) and 20 ms in c3 (bottom). D: Distribution of GABA-aetivated currents. Note main state peak at 0.8 pA. Reproduced with permission from Bormann et al. (1987). et al. (1988) for homomeric GABAA receptor com-

plexes formed by pure cc or/3 subunits expressed in oocytes. The deviation from linearity (in symmetrical CI-- concentrations) is not very conspicuous and only present at strongly depolarized holding potentials ( > + i 0 0 mV). The only other report of voltagedependent changes in the elementary GABA conductance is for bovine chromaffin cells (Bormann and Clapham, 1985). In this case, the single-channel current presents inward rectification, which is strong for only one of the three subconductance states; but the data available for the range showing rectification (positive to 0 mV) are insufficient to quantify it. On the other hand, most of the studies carried out in the mammalian CNS agree that the macroscopic

conductance evoked by GABA is outward rectifying, e.g. greater at depolarized membrane potentials (Segal and Barker, 1984a; Ashwood et al., 1987; Bormann et aL, 1987; Weiss et al., 1988; Kaneda et aL, 1989). The degree of rectification is found to be greater than that due to the asymmetry in the distribution of permeant ions (as predicted by the constant field equation) for the current induced by somatic iontophoresis of G A B A onto rat hippocampal neurones in vitro (Ashwood et al., 1987). These findings were confirmed by Bormann et al. (1987), who found significant outward rectification of GABA currents in mouse spinal neurones voltage-clamped (with the whole-cell configuration) in symmetrical CI- concentrations. Under these conditions the conductance rise

GABA RECEPTORMECHANISMS induced by 10#M G A B A increases e-fold for a 145mV depolarization. Similar results have been obtained by Kaneda et al. (1989) in acutely-dissociated rat Purkinje cells perfused intracellularly. Interestingly, these authors found that the presence of rectification for the GABA-evoked conductance depended upon the G A B A concentration tested: thus, rectification was observed at 30pM, but not at 100pM. Marked outward rectification of G A B A A receptor-induced current was also observed in oocytes expressing ~ and fl GABAA receptors (Levitan et al., 1988). The increase in G A B A slope conductance was detectable at membrane potentials positive to - 4 0 mV in cultured neurones (Segal and Barker, 1984a; Kaneda et al., 1989). In CA1 hippocampal neurones of a brain slice preparation rectification was observed at more negative potentials (about - 7 0 mV; Ashwood et al., 1987). The voltage-dependence of the rectification was not affected by changes in the reversal potential for G A B A (Segal and Barker, 1984a). The studies described above refer to the voltage-dependence of the ionic conductance activated by applications of exogenous GABA. Investigating the properties of the synaptic conductance associated with GABAergic IPSPs in mammalian CNS preparations presents considerable technical difficulties. In order to conduct such a study under voltage-clamp conditions one should reliably evoke a pure GABAAmediated synaptic event. The location of this synaptic conductance on the neurone must be such as to make it possible to achieve good voltage control of the subsynaptic membrane. Finally, in order to assess the voltage-dependence of the inhibitory postsynaptic current (IPSC), contributions by voltage-activated neuronal conductances to the electrotonic properties of the neurone and hence to the adequacy of the space-clamp must be excluded. The voltage-dependence of the GABAergic synaptic conductance has been investigated in the mammalian CNS by Barker and Harrison (1988). These authors, using whole-cell clamp in rat hippocampal neurones in culture, have reported that the peak synaptic conductance of evoked IPSCs displayed outward rectification, with a 2.5-fold increase with depolarization from - 100 to - 20 mV. Nevertheless, the voltage-dependence of the synaptic conductance did not differ significantly from that predicted by the constant field equation. The data, however, displayed considerable variability at potentials less negative than - 5 0 mV. A non-linear relation between peak synaptic current and holding potentials has also been reported by Collingridge et al. (1984) for spontaneous GABAergic IPSC in CAI neurones of the rat hippocampal slice. 3.3. KINETICSOF THE GABA A RECEPTOROPERATED E l - CHANNEL This issue was first approached by applying fluctuation analysis techniques to neurones in culture. Measuring the frequency characteristics of the current "noise" elicited by agonist application to the neuronal membrane yields an estimate of the mean open channel lifetime, provided the agonist concentration is low, e.g. the fraction of channels shut at JPN 36d--D

47

equilibrium approaches unity (Colquhoun, 1981). These studies were carried out in cultures of mouse spinal neurones (MeBurney and Barker, 1978; Mathers and Barker, 1980; Barker et al., 1982), rat hippocampal neurones (Segal and Barker, 1984a) or rat cerebellar neurones (Cull-Candy and Ogden, 1985; Cull-Candy and Usowicz, 1989). Current responses evoked by iontophoretic, pressure or bathapplied G A B A were characterized as G A B A Amediated on the basis of their dependence upon extracellular C1- concentrations (Barker and Ransom, 1978; Segal and Barker, 1984a; Cull-Candy and Usowicz, 1989) and sensitivity to bicuculline and picrotoxin (Barker et al., 1983). Most of these studies have found that the power spectra of GABA-induced current noise can be fitted by a single Lorentzian curve, with time constants in the range of 20-30 ms for mouse spinal cord neurones (McBurney and Barker, 1978; Mathers and Barker, 1980; Barker et al., 1982). The corresponding time constant for rat hippocampal neurones was found to be 22.9 ms (Segal and Barker, 1984a). Recent studies on rat cerebellar cultures have described a more complex picture: the spectrum of agonist-evoked current noise can be fitted by the sum of two Lorentzian curves with time constants of 1-2 ms and 40-80 ms at - 6 0 mV (Cull-Candy and Ogden, 1985) or 1.9 and 23.6ms at - l l 0 m V (Cull-Candy and Usowicz, 1989). Note however that noise analysis methods are limited by their frequency response: the shortest-lived open states for the GABA channel have been reported to have a time constant of 0.15-0.21 ms (see below; Mistry and Hablitz, 1989; Weiss, 1988), corresponding to cut-off frequencies around 1 kHz, which are unlikely to be resolved by noise analysis techniques. The results of noise analysis studies do not agree on the presence and degree of voltage-dependence of the time constant: Barker and coworkers have found that the time constant increased with depolarization in hippocampal neurones (by about 55% between - 6 0 to - 7 0 and + 10 mV; Segal and Barker, 1984a), but was voltage-insensitive in spinal cord cultures (McBurney and Barker, 1978; Barker et al., 1982). In cerebellar neurones only the slower of the two components was affected by voltage: its time constant increased from 23.6 to 40 ms when the membrane holding potential was depolarized from - 110 to + 50 mV (Cull-Candy and Usowicz, 1989). Another approach to the study of the kinetics of the GABAA-receptor C1- channel has been provided by the application of patch-clamp techniques to cultures of mouse spinal cord neurones (Sakmann et al., 1983; Mathers, 1985; Macdonald et al., 1989a) and chick cerebral neurones (Weiss, 1988; Weiss and Magleby, 1989). With this method, the mean open channel lifetime can be directly estimated by studying the open time distribution for a given conductance level. All authors have found that the sum of more than one exponential is required in order to fit the distribution of open times for the main conductance level. Hence, the GABA channel can exist in more than one open state with different mean lifetimes. Sakmann et al. (1983) and Mathers (1985) have reported the existence of at least two open states, while the existence of a minimum of three has been

48


proposed by Weiss (1988) and by Macdonald et al. (1989a). The presence of voltage-dependence of the channel lifetime was investigated by Weiss (1988), who found that depolarization increased the open probability (e-fold over 80 mV), but not the channel mean open time. Macdonald et al. (1989a) have described the effects of different G A B A concentrations on the kinetics of the main (27 pS) conductance state of the G A B A A channel in mouse spinal neurones in culture. In outside-out patches held at - 7 5 m V , raising the concentration of G A B A increased the frequency of channel opening and the mean open time: the latter increased from 3.7 ms at 0.5/aM GABA to 5.7 ms at 5/aM GABA. At all GABA concentrations the best fit to the open time distribution was given by the sum of three exponential functions, suggesting the existence of a minimum of three kinetically distinct open states of the GABA A channel. The shift to a longer mean open time at higher concentrations was found to be due to an increased relative frequency of occurrence of the open states with longer lifetimes, whereas the mean lifetimes of each of the three states were unaffected by concentration changes. Like other transmitter-operated channels, the GABA channel can present single openings and burst openings. In the same patch-clamp study Macdonald et al. (1989a) have analyzed the GABA channel bursting behaviour and its dependence upon agonist concentrations. The fitting of burst duration distributions indicated the presence of three populations of bursts. The shortest-lived burst component had a time constant similar to the shortest open time constant and hence probably represents a population of single openings for the shortest open state. On the other hand, the longer burst components appeared to consist of multiple openings to the longer open states. Increasing the G A B A concentration shifted burstduration frequency histograms to longer times, due to a relative increase in the occurrence of longer burst states. It is difficult to assess the impact on channel kinetics of the non-physiological environment to which the membrane patch is exposed in excised patch-clamping. In particular it is interesting to note that Weiss and Magleby (1989) observed progressive changes in the mean closed time duration of G A B A channels in patches from chick cerebral neurones in culture. These changes appear to be due to variations in the number of receptor-channel complexes available for activation (Weiss and Magleby, 1989). Another approach to studying the kinetics of a chemically gated ion channel is provided by ionic current relaxation methods. These techniques are based on measuring the time course for the attainment of a new equilibrium state after the system to be examined has been subject to a perturbation, e.g. a voltage or a concentration "jump". To our knowledge, no studies of the voltage-jump relaxation of GABA A currents have been carried out in central neurones. As for concentration jump it is technically difficult to achieve changes in the concentration of an exogenously-applied agonist that are fast enough to satisfy the assumptions of relaxation methods (Colquhoun, 1981). A possible solution to the problem of obtaining fast changes in agonist concen-

trations is to use the synaptic release of the transmitter which normally activates the channel to be studied and measure the decay of the postsynaptic current induced by such spontaneous or stimulusevoked synaptic release. Provided that transmitter concentration at the synaptic cleft decreases to zero fast enough, no transmitter rebinding to the receptors should occur during the decay phase, and therefore once a channel closes during the decay, it will not open again. Under these conditions the decay of the synaptic current will reflect the time course of channel closure, in other words the mean channel lifetime. Several authors have investigated the decay of GABAergic inhibitory postsynaptic currents (IPSCs) in slice or culture preparations of the rat hippocampus (Collingridge et al., 1984; Segal and Barker, 1984b; Barker and Harrison, 1988). The decay of GABAergic IPSCs in rat hippocampal neurones was found to be monoexponential both for spontaneous (Collingridge et al., 1984) and evoked IPSCs (Segal and Barker, 1984b; Barker and Harrison, 1988). While application of tetrodotoxin or Cd 2+ reduced the amplitude and frequency of spontaneous IPSCs, it failed to affect the time course of the resulting "miniature" IPSCs (Collingridge et al., 1984). The decay time constant for GABAergic IPSCs was found to be temperature-dependent, being faster at higher temperatures (Collingridge et al., 1984), a characteristic similar to that observed for end-plate currents at the neuromuscular junction (Anderson and Stevens, 1973). The Q10 value was 3.3 for the decay time constant at 0 mV (Collingridge et aL, 1984). The results described below will therefore refer to room temperature. All authors have found that the decay of IPSCs is slower at depolarized membrane potentials. An efold increase in the decay time constant over a 146 mV change in holding potential was reported by Collingridge et al. (1984) for CA1 neurones in the rat hippocampal slice. In hippocampal cultures the IPSC decay time constant approximately doubled when the membrane potential was depolarized from the resting value ( - 5 0 to - 6 0 m V ) to a holding potential 10-20mV positive to the E~psp which under these conditions was - 1 5 to 0 m V (Segal and Barker, 1984b). An increase in decay time constant with depolarization was also observed in most of the neurones tested in hippocampal cultures by Barker and Harrison (1988) with whole-cell patch-clamp. In this study a considerable variability in the time constant values was however apparent, with a range of 10-40ms at - 4 0 m V . In the same preparation Segal and Barker (1984b) had found a time constant value of 20.5 ms at - 5 0 - 6 0 mV with two electrode voltage-clamp in the presence of intracellular Cs + (in order to block voltage-dependent K + conductances). Collingridge et al. (1984) found a decay time constant of 19 ms at 0 mV (extrapolated value at - 6 0 mV about 13 ms), therefore at the lower end of the range described by Barker and Harrison (1988). Markedly different results have been obtained by Weiss et al. (1988) in chick cerebral neurones in culture with whole-cell patch-clamp (in the presence of intracellular Cs +). These authors observed that the decay phase of the spontaneous GABAergic IPSC was monoexponential only in two-thirds of events.

GABA RECEPTORMECHANISMS The time constant for these single exponential decays was voltage-dependent, increasing from 15ms at - 8 0 m V to 50ms at + 4 0 m V . After exposure to tetrodotoxin, Cd 2÷ or low Ca 2+ media, the IPSCs were smaller in amplitude and consistently showed a monoexponential decay. Surprisingly, the time constant for these "miniature" IPSCs no longer increased with depolarization and was slightly faster than in control conditions. Weiss et al. (1988) have also shown the existence of a positive correlation between peak IPSC amplitude and decay time constant: both Collingridge et al. (1984) and Barker and Harrison (1988) have investigated this point (a negative correlation would be expected if the space-clamp was not adequate; see Johnston and Brown, 1983) and have not observed any correlation between the two parameters. The positive correlation described by Weiss et al. (1988) has been interpreted by the same authors as evidence of significant rebinding of the transmitter to the receptor-channel complex during IPSC decay. The divergence between this study and that on rat hippocampal neurones showing similar decays for normal and "miniature" IPSCs (Collingridge et al., 1984) could be due to differences in the time course of changes in G A B A level in the synaptic cleft. It is conceivable that differences in the geometry of the GABAergic synapse and in the number of glial cells present may affect the rate of transmitter elimination from the cleft. It is, however, more difficult to explain the discrepancy in the voltage-dependence of the time co~istant: on the basis of their results, together with single-channel records (Weiss, 1988), Weiss et al. (1988) have suggested that the voltage-dependence may reside in the rate constants for the binding of G A B A to the receptor rather than in the channel open time. It is difficult to draw a coherent picture of the kinetics of the G A B A A receptor-operated channel from the data obtained with studies of agonist current fluctuations, single-channel currents and IPSC decay in view of the many experimental variations in published studies. On the basis of their single-channel recording data from mouse spinal neurones in culture, Macdonald et al. (1989a) have proposed the following working model of the kinetics of the GABAA receptor-channel complex: 2A+R~-A+AR~A

11,

2 R ~ A 2R'

11,

Closed

states

Open

states

11,

A + AR* ~ A 2R* ~ A2R'*

where A is the agonist, R the receptor complex, R' an additional conformation of the same and * indicates open states. The model is consistent with the observation of at least three open and three closed states. The authors have suggested that the monoliganded open channel corresponds to the shortest-lived open state. An increase in agonist concentrations would cause a shift towards the double liganded states and, as actually observed, an increase in the relative frequency of the longer open states. No attempt was however made to test the model by estimating rate constants, calculating the predicted probability density function and comparing it with the observed open and closed time distributions. Such an investi-

49

gation was carried out by Weiss and Magleby (1989) on chick cerebral neurones. These authors found that, since three open and three closed states were observed, at least five of the several possible six-state mechanisms considered could fit the data (e.g. sets of rate constants could be found for these schemes which gave probability density function consistent with the open and closed time distributions). These authors discovered the existence of a correlation between adjacent closed and open times: the longer openings were associated with the shorter closures. This observation indicates that, for the G A B A channels, the connectivity of open and closed states is greater than one, namely there is more than one gateway state for the transition between closed and open states. Given this further constraint, only one of the proposed mechanisms could be fitted with rate constants such as to describe the data adequately: the other four had to be discarded, some because they had a single gateway state and some because the fitted rate constants, which could account for the inverse correlation between adjacent intervals, implied a modal behaviour of the channel (e.g. the switching of the channel between two different values of mean open times), which contrasted with the experimental observations. The remaining possible scheme is the following: C 6 ~ C 5 ~ - C4

11,

It

03

02 ~- O1

where C4-C6 and O1-O3 represent discrete kinetic states. Note that the binding steps for the agonist are not designated. 3.4. CHARACTERISTICSOF GABAA-ACTIVATED CI- CHANNELS 3.4.1. Agonists

By definition, G A B A g agonists are able to open the anion channel associated with the GABA g receptor complex when they bind to the recognition site. The properties of the resulting elementary current have first been shown to be dependent upon the nature of the agonist by Barker and Mathers (1981). Fluctuation analysis of matched current responses to GABAA analogues in mouse spinal neurones in culture indicated that the macroscopic time constants of C l - channels activated by the agonists differed significantly from that of GABA, whereas the single-channel conductance was similar for all agonists at 15.4--17.9 pS. At holding potentials between - 6 0 and - 7 0 m V the average duration of opening ranged between 5.5 and 76.3 ms and appeared to be inversely correlated with the potency displayed by each agonist in displacing bound G A B A from rat brain membranes. Thus, the longest channel duration was observed for muscimol and the shortest for imidazole acetic acid, while GABA-evoked openings had a somewhat intermediate duration (30.4 ms). These results were confirmed by Segal and Barker (1984a) with fluctuation analysis in rat hippocampal neurones in culture with reported time constants of 22.9 ms and 38.8 ms at - 7 0 mV for G A B A and muscimol, respectively.

50

L. S1VILOTTIand A. NISTRI

The advent of single-channel recording has not yet cast further light on this point. In outside-out patches from chick cerebral neurones in culture, Mistry and Hablitz (1989) have shown the presence of two populations of openings for the main subconductance state of the GABA channel. The time constants for the two exponentials describing the open time distribution were similar for GABA (0.15 + 0.04 and 2.07 + 0.37 ms) and isoguvacine (0.48 + 0.22 and 2.21 + 0.43 ms). The situation was different for muscimol-evoked openings, since the best fit to the open-time distribution required three exponential terms (time constants 0.16 + 0.08, 1.15 _ 0.43 and 6.53 + 4.07 ms). Given the scarcity of experimental work on this issue, it is hard to pinpoint the reason for the discrepancy between the results of Barker and co-workers and those of Mistry and Hablitz (1989). Apart from the difference in species, a possible explanation lies in the differences in the techniques used (see Section 3.3). It must be said that the dependence of channel kinetics upon agonist structure is a feature of other ligand-gated channels, most notably the acetycholine nicotinic receptor (Katz and Miledi, 1972; Colquhoun et al., 1975; Auerbach et al., 1983; Colquhoun and Sakmann, 1985). 3.4.2. Antagonists As shown by functional and radioligand binding studies, the antagonistic effects of bicuculline and picrotoxin on GABAA responses are mediated by distinct sites on the GABA A receptor complex. In most experimental models, while bicuculline behaves as a competitive GABA antagonist acting on the recognition site, picrotoxin appears to bind to a different site, which overlaps at least in part the site for the binding of TBPS and pentylenetetrazole (see Section 2.2.3). Indeed, picrotoxin does not block GABA responses competitively; furthermore 10 ~M picrotoxin, but not bicuculline, antagonizes GABA currents when applied intracellularly to isolated internally perfused frog dorsal root ganglion neurones (Akaike et al., 1985). In the same cells, the outward rectification of GABA-evoked currents (observed even at hyperpolarized membrane potentials) was reduced by bath application of 10 #M picrotoxin, but not of bicuculline (Akaike et al., 1985). This observation would suggest that picrotoxin blocks the ionic channel activated by GABA; it must however be noted that this type of mechanism---e.g, voltage-dependent channel block--is usually seen after channel block by charged molecules, whereas picrotoxinin, the active component of picrotoxin, is neutral. Barker and co-workers (1983) have investigated the effects of bicuculline, picrotoxin and pentylenetetrazole on the properties of GABA-activated channels in mouse spinal neurones in culture. Pressure-applied bicuculline or picrotoxin failed to affect single-channel conductance and mean open time as determined by fluctuation analysis of GABA currents under two electrode voltage-clamp conditions. Only pentylenetetrazole was found to alter the power spectrum of GABA-evoked fluctuations, producing a decrease in channel open time in most of the cells tested. Similar conclusions concerning the mechanisms of picrotoxin antagonism were drawn by Segal and Barker (1984b),

who found that, in rat hippocampal neurones in culture, local application of 100 # M picrotoxin had no effect on either the power spectrum of the current variance evoked by G A B A or the single-channel conductance. Picrotoxin did decrease the peak amplitudes of evoked GABAergic IPSCs in this preparation, but left the IPSC time course unchanged. It would therefore appear that those G A B A channels which open in the presence of picrotoxin maintain their normal kinetics. In mouse spinal neurones in culture, the question of the effect of picrotoxin on the elementary GABA conductance has been approached with single-channel recording techniques by Twyman et al. (1989a). In outside-out patches held a t - 7 5 mV these authors found that pressure-ejected 10/~M picrotoxin did not alter the single-channel conductance in the presence of 2#M GABA. Contrary to the observations of Barker et al. (1983), picrotoxin was found to affect the kinetic properties of the GABA channel. Indeed, in the presence of picrotoxin, both the mean open time and the frequency of channel opening were reduced. The analysis of the frequency distributions of the burst duration and of the number of channel openings per burst showed that the action of picrotoxin consisted of a shift towards shorter burst durations (from 12.9 to 7.5ms) associated with a decrease in the mean number of openings per bursts from 2.01 to 1.55. The same study has also examined the effect of picrotoxin on GABA-evoked whole-cell currents. Apart from a decrease in the neuronal leak conductance, 10/~M picrotoxin reduced both the inward current and the conductance increase evoked by GABA, albeit to a different extent (i.e. the conductance change was more affected). It would be important to clarify whether the effects of picrotoxin are voltagedependent by investigating the current-voltage relationship under the different experimental conditions and in particular to establish the behaviour of the picrotoxin antagonism of G A B A at holding potentials at which the GABA-evoked current response inverts its polarity from inward to outward. Penicillin is known to antagonize GABAA-mediated responses with a non-competitive mode of action (Macdonald and Barker, 1977; Pickles and Simmonds, 1980). In excised outside-out patches from mouse spinal cord neurones in culture, Chow and Mathers (1986) have shown that pressure-applied penicillin (2 mM) reduced the mean open time of single channels activated by pressure-applied GABA (1/~M). This decrease in mean open time (from 2.7 to 0.92 ms at - 80 mV) was due to a decrease in the time constant of the longer state from 4.29 to 1.12ms. Shorter openings (with a mean lifetime of about 0.4 ms) were unchanged. Lower penicillin concentrations (0.2 mM) did not significantly affect GABAinduced currents. These findings are not inconsistent with an open channel block mechanism for the convulsant effect of penicillin. 3.4.3. Modulators 3.4.3.1. Barbiturates These are known to exert at least two separate actions on the GABA receptor-channel complex: an

GABA RECEPTORMECHAImSMS enhancement of the effects of GABA A agonists and a direct channel opening action (when moderate to high concentrations of barbiturates are applied in the absence of GABA agonists). The membrane mechanism of the latter effect was first described by Mathers and Barker (1980) by the application of current fluctuation analysis to mouse spinal neurones in culture. At - 5 0 to - 9 0 m V both GABA and (-)pentobarbitone (the latter applied by pressureejection from pipettes containing a 100/aM solution) elicited an inward current associated with an increase in membrane current variance. The channel openings evoked by the two compounds had a similar elementary conductance, but different lifetimes: the mean open time for pentobarbitone-activated channels was approximately five-fold longer than that of GABAactivated channels (153.4 vs 30.7 ms, respectively). It is unlikely that this effect resulted from the enhancement by pentobarbitone of the actions of synaptically-released ambient GABA, since the experiments were carried out in the presence of high Mg 2÷ and tetrodotoxin. Furthermore, power spectra of control current fluctuations (in the absence of exogenous compounds) were linear, indicating that the contribution of endogenous GABA was too small to be resolved by the technique and hence that the ambient GABA concentration must have been very low. While pentobarbitone could conceivably increase the levels of extraceilular GABA by acting on uptake and/or release mechanisms (Aickin and Deisz, 1981), the authors have suggested that the data could be explained more simply by a direct effect of the drug on the anion channel. The same group (Barker and McBurney, 1979; Study and Barker, 1981) has been the first to investigate the barbiturate potentiation of GABAA responses in terms of changes in channel conductance and kinetics. Again, no effect of barbiturates on the elementary conductance of the GABA-activated channel could be detected, but the enhancement of GABA current responses appeared due to changes in the mean open time of the channel, hence in its kinetics. In fact, the mean lifetime of the open channel in the presence of GABA increased approximately 3-fold following passive diffusion of pentobarbitone (100/aM; Study and Barker, 1981) and from 24 to 120ms after iontophoresis of phenobarbitone (Barker and McBurney, 1979). In the latter study, phenobarbitone greatly slowed the decay of a population of spontaneous GABAergic synaptic potentials, thus corroborating the hypothesis of a lengthening of the channel time constant by barbiturates. The magnitude of the pentobarbitone potentiation of the macroscopic GABA current responses expected on the basis of the change in mean open time was, however, greater than the one observed experimentally. Study and Barker (1981) inferred that the action of pentobarbitone on mean open time must be in part counteracted by a concurring reduction in the frequency of GABA channel opening. Similar results were obtained in rat hippocampal neurones in culture by Segal and Barker (1984b): application of 100/~M pentobarbitone increased by 132% the macroscopic time constant of opening calculated from the GABA power spectrum. The drug also substantially prolonged (by 156%) the time constant

51

for the monoexponential decay of evoked GABAergic IPSCs. The peak amplitude of the latter was unchanged. Pentobarbitone (bath-applied at 100/~M) also slowed by more than 4-fold the monoexponential decay of GABAergic spontaneous IPSCs recorded from voltage-clamped CAI neurones in a rat hippocampal slice preparation (Collingridge et al.,

1984). Single-channel recording has confirmed some of the findings of the earlier studies. The greater resolution allowed by this technique has made it possible to establish the effect of barbiturates on the relative contribution to macroscopic GABA currents by single vs burst openings of the channel (Macdonald et al., 1989b). In this study, carried out on mouse spinal cord neurones in culture, phenobarbitone and pentobarbitone (pressure-ejected solutions 500 and 50/~m, respectively) did not affect single-channel conductance values (27 and 16.5pS) or the relative frequencies of opening to the two more common subconductance states. In the presence of barbiturates profound changes in the kinetics of the main subconductance state (27 pS) were observed. The mean open time evoked by 2 # u GABA (3.5 ms in control conditions) rose to 5.4 and 8.3 ms in phenobarbitone and pentobarbitone, respectively, and the proportion of time spent in the open state increased from 5.6 to 7.5 and 12.5%, respectively. This effect was due to a shift in the relative frequency of occurrence of the different open states of the GABA channel: in the presence of barbiturates, the time constant of the three open components was unchanged, but the longest-lived open state (03) occurred more frequently than in control conditions, whereas the shortest component (Oj) became infrequent, virtually undetectable. Indeed, the relative area of the longest component 03 in the open time distribution was raised from 0.24 to 0.59 and to 0.80 by phenobarbitone and pentobarbitone, respectively. Closed state kinetics were unaffected by barbiturates. The channel burst behaviour was deeply affected, with an increase in mean burst duration from 10.0 to 14.1 and to 21.4ms and in the percentage of time spent in a burst state from 6.9 to 8.5 and to 8.8% in phenobarbitone and pentobarbitone, respectively. Burst frequency was slightly decreased (from 6.7 to 5.4-5.6 per s). The analysis of the frequency distribution of burst durations showed that the observed increase in mean burst duration was due to an increase in the time constant of the longest lived of the three burst components from 27.5 to 35.7 ms in phenobarbitone and to 43.1 ms in pentobarbitone. This was associated with an increase in the relative frequency of such a burst component, manifest as an increase in its relative area from 0.21 to 0.30 in phenobarbitone and to 0.44 in pentobarbitone. The frequency of the two shorter burst components was proportionally decreased. These authors point out that the longer burst components are constituted by multiple openings to the two longer open states 02 and O3: therefore, one of the reasons for the increase in the lifetime of the longest burst component, in the presence of barbiturates, must be the increased frequency of the longest open state O3. Macdonald et al. (1989b) predicted that the burst lifetime prolongation should also be associated with an increase in the

52


number of openings per burst. This increase was observed by Twyman et al. (1989a). In their study, pressure-application of a 50#M pentobarbitone solution increased the mean number of openings per burst from 2.01 to 2.39, while leaving the mean intraburst closed time unchanged. These data can be seen to be quite consistent with the results of fluctuation analysis when the low frequency response of the latter is taken into account. Indeed, it is likely that the single Lorentzian component observed in the G A B A power spectrum corresponds to the longer channel bursts. The enhanced probability of channel opening to the longest open state induced by barbiturates is a distinct effect from the one following an increase in the GABA concentration, which augments the frequency of the two longer open states O2 and O3 vs the shortest O1 (see Section 3.3; Macdonald et al., 1989a). As pointed out by Macdonald et al. (1989b), the complexity of kinetic models for GABA receptor channel gating makes it difficult to establish which rate constant(s) may be altered by barbiturates. In fact, while it is likely that the rate constants involved are those which regulate entry into the different open states, such a site for the effect of barbiturates should imply parallel changes in the time constants of the closed states directly connected to the open states, changes that have not been detected so far. 3.4.3.2. Benzodiazepines Unlike barbiturates, the benzodiazepine flunitrazepam (1/~r~) does not appear to activate GABA C1- channels per se in outside-out patches of rat neocortical neurones in culture (Vicini et al., 1987). Higher doses or different benzodiazepines have not been tested. Benzodiazepines do potentiate GABA responses via a mechanism which was first studied with current fluctuation analysis (Study and Barker, 1981). These authors recorded current responses to iontophoretic applications of G A B A in control conditions and in the presence of diazepam (diffusing from a pipette containing a 17/~r~ solution) from mouse spinal neurones under two-electrode voltageclamp conditions. Diazepam did not affect the singlechannel conductance activated by GABA and produced a fairly small increase ( + 3 2 % ) in the channel mean open time. The modest size of this effect was insufficient to account for the marked potentiation of the GABA-induced current observed in the presence of the benzodiazepine. Hence a significant component of this enhancement must be due to an increase in the frequency of opening of the channel. Similar results were obtained by Segal and Barker (1984b) on rat hippocampal neurones in culture: pressure-applied diazepam (10 # M) increased by 30% the mean open time of the GABA-operated channel as estimated from the cut-off frequency of the power spectra of current size elicited by G A B A or muscimol. Segal and Barker calculated that the frequency of channel opening would have to increase by 39% in diazepam to explain the degree of enhancement of GABA currents. In the same preparation, the amplitude of GABAergic IPSCs was also greatly increased by diazepam. The benzodiazepine slowed the IPSC decay, increas-

ing its time constant by 52% (Segal and Barker, 1984b). With the reservations outlined in Section 3.3, this finding confirms that diazepam prolonged the mean open time of the G A B A channel. These results have been confirmed by single-channel recording. In outside-out patches from primary cultures of mouse spinal neurones application of diazepam doubled the frequency of burst openings to the 27pS subconductance state evoked by 2/tM GABA. This effect was associated with an increase in the channel mean open time from 5 to 7.3 ms. No changes in single-channel conductance or in the lifetime of each of the different open states was observed (Twyman et al., 1989b). Similarly, Vicini et al. (1987) found that, in outside-out patches from rat cortical neurones in culture, flunitrazepam increased the frequency of opening of the GABA channel (by 40%) and the relative frequency of the longer component of bursts (from 37 to 60%). It must be noted that in this study 2/~M bicuculline was added when flunitrazepam was applied to the patches in order to reduce the frequency of multiple channel openings (bicuculline was not present during control G A B A applications). These authors also pointed out that a low, yet suprathreshold, concentration of GABA was present in the patch environment in control conditions, possibly because of release of GABA by the culture. In these experiments the Ca 2+ : Mg 2+ ratio was 1 : 1, i.e. different from the 1 : 6 or 1 : 10 ratios chosen by Macdonald et al. (1989a) and Mathers and Barker (1980), respectively. Earlier reports from single-channel experiments in mouse spinal neurones suggested that diazepam increased the burst duration (Redmann and Barker, 1984; Rogers and Macdonald, 1986). These findings have not been Confirmed so far. As far as the effects of benzodiazepine antagonists and inverse agonists on the G A B A channel properties are concerned, the only available information comes from the work of Vicini et al. (1987) on outside-out patches from rat cortical neurones in culture. These authors found that the antagonist flumazenil (diffusing from a pipette containing a 5 #M solution) had no effect, whereas D M C M (same concentration and method of application) reduced the frequency of channel opening induced by 1/~M GABA to 40% of control and decreased the relative frequency of occurrence of the longer burst events from 37 to 25% of the total. 3.4.3.3. Steroids Barker et al. (1987) have investigated the action of the steroid anaesthetic, alphaxalone, on rat spinal neurones in culture under two-electrode voltageclamp or whole-cell clamp. Pressure-applications of low alphaxalone concentrations (I/~M) in the presence of GABA were found to cause a marked increase in the mean open time of the GABA-activated channel (from 30 to 74 ms at - 5 0 mV). The steroid did not significantly change single-channel conductance. At higher concentrations (10 pM), alphaxalone elicited a bicuculline-sensitive current per se, which had the same reversal potential as that evoked by GABA. The cut-off frequency of the power spectrum for alphaxalone responses in the absence of GABA

GABA RECEPTORMECHANISMS indicated a much longer mean open time (105 ms on average) that that observed for GABA responses. In 2/4 neurones a good fit of the alphaxalone power spectrum required an additional Lorentzian function, suggesting the presence of a class of channel openings with an appreciably shorter lifetime (5 ms). Cottrell et al. (1987) have reported that, in outsideout membrane patches from bovine chromaffin cells, bath-applied 10/~M alphaxalone elicited the opening of single channels with a conductance similar to that of channels opened by 1/~M GABA (29 and 32 pS, respectively). The whole-cell current induced by alphaxalone per se showed outward rectification, was sensitive to bicuculline and phenobarbitone and reversed at the same membrane potential as that produced by GABA.

4. GABAe RECEPTORS The classification of GABA receptors into two major categories, A and B, originates from the demonstration (Hill and Bowery, 1981) that fl,p-chlorophenyl-GABA (baclofen), a GABA receptor agonist able to cross the blood-brain barrier, binds selectively to a population of GABA recognition sites (termed B). GABAA receptors are therefore considered all those insensitive to baclofen and possessing the biochemical and pharmacological properties (including barbiturate and benzodiazepine binding sites) described in the preceding Sections 2 and 3. 4.1. BIOCHEMICALCHARACTERISTICS AND AUTORADIOGRAPHIC DISTRIBUTION

The properties of GABAB receptor-mediated responses in peripheral tissues have been recently reviewed (Bowery, 1989). In the central nervous system synaptie membranes usually present two classes of binding sites for baclofen (Hill and Bowery, 1981; Falch et al., 1986). GABA and (-)baclofen have an approximately similar potency (around 100riM) in terms of binding activity to the high affinity site while 3-aminopropylphosphinic acid is 100-fold more active. Several GABAA receptor agonists are either at least 100 times weaker as ligands for the GABAB sites (e.g. muscimol or 3-aminopropanesulphonic acid) or are virtually inactive (e.g. isoguvacine, imidazoleacetic acid). Bowery et al. (1983) have observed two unusual characteristics of GABA a binding, namely its strong reduction by the membrane detergent Triton X-100 (rather than enhancement via the unmasking of recognition sites) and the absolute requirement for Ca 2+ (or Mg2+; up to 2.5 raM) (contrast this with the inhibitory action of intracellular Ca 2+ on GABA A receptor-mediated responses; InDue et al., 1986). As far as GABAa receptor antagonism is concerned, both picrotoxin and bicuculline are ineffective (Hill and Bowery, 198 l). A weak competitive antagonism (pA: about 4) is produced by phaclofen (Kerr et al., 1987) while its derivative 2-hydroxysaclofen seems to be somewhat more potent (pA 2 = 5; Kerr et al., 1988). A more powerful antagonism might be obtained with 5-methoxybenzo[b]furan-2-ylbutanoic acid (Beattie et al., 1989) although its limited water solubility can restrict its use

53

for in vitro experiments. Non-selective antagonism towards both GABAA and GABAB receptors is produced by 6-aminovaleric acid (Robinson et al., 1989). The isolation and purification of GABAB receptors are currently at a preliminary stage, owing no doubt to the lack of very potent and selective iigands. It will thus be interesting to compare the structure of GABAA and GABAB receptors in order to derive even more powerful and specific agents for these sites. By exploiting the Ca 2+ dependence and the insensitivity to isoguvacine of GABA8 sites, Bowery et al. (1987) have mapped the distribution of [3H] GABA binding to GABA B sites in the rat central nervous system. In most areas there is a predominance of GABAA sites over GABAB ones, although apparent exceptions to this rule are the molecular layer of the cerebellum and the interpeduncular nucleus of the mesencephalon. The functional significance of various ratios of GABAA over GABAB sites is still unclear, since at least some of these sites may be extrasynaptic and their absolute distribution to preor postsynaptic membranes is not firmly established. 4.2. CELLULARMECHANISMSUNDERLYING RESPONSES MEDIATED BY GABA a RECEPTORS Prior to the characterization of GABA a receptors, it was shown that baclofen has a neurodepressant activity in the spinal cord (Pierau and Zimmermann, 1973; Davidoff and Sears, 1974) and in various brain areas (Curtis et al., 1974; Davies and Watkins, 1974). Unexpected characteristics of the action of baclofen were its insensitivity to bicuculline antagonism and the slow and generally sustained nature of the inhibition (unlike the rapid action of GABA itself). Intracellular studies on cat motoneurones demonstrated how the inhibitory effect of baclofen was associated with little change in membrane potential or conductance (Pierau and Zimmermann, 1973; Fox et al., 1978). Although an interaction of baclofen with the postsynaptic receptors for various transmitters has been argued (see for example Davies, 1981), it was generally suggested that the main effect of baclofen was to inhibit presynaptically the release of excitatory transmitters (Fox et al., 1978). In keeping with electrophysiological results, biochemical studies also indicated that baclofen reduces the Ca 2+ -dependent release of acetylcholine (Nistri, 1975) or of glutamate and aspartate (Potashner, 1979; Collins et al., 1982), even if the concentrations of baclofen used were comparatively larger (in the high micromolar range) than those employed for binding data (for a review of baclofen-induced depression of peripheral transmitter release, see Bowery, 1989)~ An important clue for the understanding of the presynaptic action of baclofen was provided by the demonstration that this drug reduced the slow Ca 2+ current of dorsal root ganglion neurones in the chick (Dunlap, 1981) and in the rat (Dolphin and Scott, 1986). In analogy with these observations on the cell body of neurones, it was inferred that baclofen may depress transmitter release from presynaptic endings by impairing the Ca 2+ mechanisms necessary to elicit vesicular exocytosi~ How these studies can relate to the physiological, GABA-mediated presynaptic inhibition in the spinal cord is not completely clear (see also Section

54

L. SIVILOTTIand A. NISTR!

7.11.2), as presynaptic inhibition is typically blocked by the GABAA antagonists, bicuculline and picrotoxin (Nistri and Constanti, 1979). Nevertheless, GABAa receptor mechanisms have been detected on A6 and C neurones in dorsal root ganglia but not on the faster conducting Aft cells (D6sarmenien et al., 1984; Schlichter et al., 1987) and can perhaps contribute to a control of signals in moderately fast or slow afferents from the periphery. The biochemical link between baclofen binding and Ca 2+ current depression has also received much attention. It is clear that a GTP-binding protein (G protein) is an essential intermediary in this phenomenon (Holz et al., 1986; Dolphin and Scott, 1987): in particular, the G protein activity is specifically inhibited by pertussis toxin, which, in low concentrations, also abolishes the effect of baclofen. Although intracellular second messenger systems are often stimulated by neurotransmitter mechanisms as a consequence of their activation of a G protein, in the case of rat dorsal root ganglion cells it seems that the G protein operates as a direct coupler between GABAB receptors and Ca 2+ channels (Dolphin et al., 1989). In sharp contrast to the studies of baclofen-induced suppression of Ca 2+ currents, recordings from rat hippocampal neurones in vitro have clearly shown that baclofen has a direct hyperpolarizing action on the postsynaptic membrane via activation of a K + conductance (Newberry and Nicoll, 1984a, 1985). This effect of baclofen seems to be more prominent on the pyramidal cell dendrites, an observation which demands caution in interpreting data on reversal potentials as those remote regions may not be fully controlled by a somatic microelectrode. The hyperpolarizing action of baclofen is blocked by Ba 2+ or by phaclofen (Dutar and Nicoll, 1988a) and may find a physiological correlate in a late IPSP evoked by orthodromic electrical stimulation (Fig. 2). An interesting property of the baclofen-induced response is its anomalous dependence on the cell membrane potential (Newberry and Nicoll, 1985): when the cell is depolarized away from the K + equilibrium potential, the response amplitude declines. Paradoxically this phenomenon implies that the postsynaptic inhibitory action of baclofen is progressively less efficient preGontrol

cisely at the stage when it is more useful as the neurone becomes more excitable. Analysis of GABAinduced responses on the same neurones (Newberry and Nicoll, 1985) shows that they are very complex in the dendrites, comprising a depolarization and a fast hyperpolarization both caused by GABAA receptor activation plus a slow hyperpolarization attributable to G A B A B receptors. A more direct approach to the postsynaptic action of baclofen has been obtained with voltage-clamp studies on hippocampal neurones in culture (G~ihwiler and Brown, 1985): baclofen has been found to elicit an outward K + current which displays strong inward rectification (i.e. depression at more depolarized membrane potentials) and is blocked by Ba 2+ and Cs +. Interestingly, the same investigation has reported that the effects of GABA via GABA B receptors were minimal when compared with the GABAA receptor-mediated responses. The issue of strong rectification of the baclofenelicited responses is apparently complicated by regional variabilities. For example, in the same animal species, i.e. the rat, the current-voltage relation for neurones in the locus coeruleus or in the dorsal horn is nearly linear from values slightly positive to the K + equilibrium potential (Osmanovi6 and Shefner, 1988; Allerton et al., 1989). Although Ba 2+ or Cs + could block the postsynaptic effect of baclofen on these neurones, perhaps suggesting that this drug activated K + currents similar to those found in the hippocampus, the hyperpolarization induced by baclofen (or the underlying conductance increase) was not substantially depressed by making the cell membrane potential less negative. It is not yet clear whether these differences can be attributed to some distinct properties of K + channels in these neurones. Should this be the case, it would imply that the control by GABAB receptor activation of neuronal excitability shows significant regional variations. Further studies on hippocampal neurones have focussed on the subcetlular mechanism(s) responsible for the baclofen-induced activation of a K + conductance. Andrade et al. (1986) have found that the action of baclofen is linked to the activation of a G protein, as the response could be prevented by

PhecloTen

I

-.-~

Wssh

~

l 3~ms

:.f

i,

FIG. 2. IPSPs (fast and slow) of a pyramidal neurone in rat hippocampus in vitro. Top records show brief EPSP followed by large hyperpolarizing potential comprising a fast and a slow component; the latter is selectively blocked by phaclofen (compare also superimposed records in right panel). Bottom records show EPSP followed by fast IPSP only (stimulation intensity is now reduced). The fast IPSP is not antagonized by phaclofen. Reproduced with permission from Dutar and Nicoll (1988a) (Nature Vol. 332, pp. 156-158. Copyright © 1988 Macmillan Magazines Ltd).


pretreatment with pertussis toxin or modified by injecting intracellularly various guanine nucleotides. The G protein activity is in turn regulated by phosphorylation through an intracellular protein kinase. In analogy to the studies on dorsal root ganglia reported earlier, the G protein mechanism apparently does not rely on the formation of intracellular second messengers but is directly linked to the function of the K + channels (Andrade et al., 1986). The neurotransmitter 5-hydroxytryptamine, which operates through a distinct class of receptors, mimics the action of baclofen, with which it shares a common effector system based on the link between G protein and K ÷ channel activity (Andrade et al., 1986; Dutar and Nicoll, 1988b). The excitatory amino acid agonists kainate and quisqualate block the K ÷ conductance system activated by baclofen or 5-hydroxytryptamine (Rovira et al., 1990). An important, yet unsolved, problem is the possible heterogeneity of GABA B receptors. For sake of simplicity such a problem can be divided into various questions: (1) are the effects of baclofen and those of GABA on GABAs receptors identical? (2) can baclofen act on more than one type of GABA Breceptor? (3) are the pre- and postsynaptic actions of baelofen mediated by a similar receptor coupled to two distinct effector mechanisms (i.e. Ca 2÷ current depression and K ÷ current activation, respectively)? In guinea pig hippocampal neurones in vitro there are important differences in the effects of GABA and baclofen after pharmacological block of GABA A receptors. In particular three findings are of special interest: the inward rectification for GABA is much less than that for baclofen, 4-aminopyridine blocks the effect of baclofen but not that of GABA and, finally, the depression of neurally-evoked EPSPs is far greater with GABA than with baclofen (Ogata et al., 1987). Furthermore, very small concentrations of the cholinergic agonist carbachol (by itself ineffective on the cell membrane properties) significantly depress the action of baclofen but not that of GABA (Mtiller and Misgeld, 1989). This issue is also complicated by the relatively complex direct actions of the GABAB antagonist phaclofen on intrinsic voltagedependent conductances, so that it is difficult to use this substance simply to block selectively GABA a receptors (Mfiller and Misgeld, 1989). It has been suggested that the postsynaptic hyperpolarization produced by baclofen may be due to activation of anomalous rectifier channels similar to those blocked by the excitatory peptide substance P (Ogata et al., 1987). GABA would probably be far less selective (or potent) than baclofen in opening channels: hence, in order to control neuronal excitability, any late inhibitory postsynaptic action of GABA additional to the one mediated by GABAA receptors would probably rely more on separate mechanisms (perhaps of metabolic nature; see Corradetti et al., 1987) than on activation of GABAB receptors. Studies of other central neurones, e.g. in the lateral geniculate, also show that the inhibition of neuronal firing by a late IPSP mediated via GABA e receptors is rather weak when compared to other inhibitory processes (Soltesz et al., 1989a). In keeping with these results recent in vivo studies on cat motoneurones have confirmed that the postsynaptic

55

action of baclofen is practically negligible when compared to its presynaptic effects (Edwards et al., 1989). Even if postsynaptic GABAe receptors seem to have a comparatively small physiological role, it is feasible that they are heterogenous. Biochemical studies (reviewed by Bowery, 1989) have found contrasting effects of baclofen on the synthesis of cyclic AMP (note however that electrophysiological studies exclude any major action of baclofen on adenylcyclase activity; Andrade et al., 1986; Dolphin et al., 1989) which might be accounted for by distinct receptor classes. Intracellular recordings have indicated that baclofen can reduce synaptic transmission at pre- and postsynaptic sites (Inoue et al., 1985; Allerton et al., 1989). The pharmacology of these responses is quite dissimilar since the presynaptic effect is not blocked by phaclofen or by pertussis toxin (both of which abolish the postsynaptic responses) but is antagonized by phorbol esters, agents able to activate protein kinase C (Dutar and Nicoll, 1988b). On rat motoneurones, too, the presynaptic depressant action of baclofen is insensitive to phaclofen (Wang and Dun, 1990). These results indicate that the receptors as well as the effector systems for baclofen may differ depending on their pre- or postsynaptic location. Unfortunately, the small size of the presynaptic fibre endings responsible for transmitter release is not suitable for standard electrophysiologicai techniques, hence further progress in our understanding of this important issue is necessarily slow. Finally, both baclofen and GABA strongly depress IPSPs (see for instance Inoue et al., 1985; Ogata et al., 1987): this phenomenon presents considerable interest since it involves autoregulation of endogenous GABA release, which will be discussed in the separate section below.

5. AUTOREGULATION OF GABAERGIC TRANSMISSION While GABA is regarded as the major inhibitory neurotransmitter in the brain, some characteristics of GABA-mediated transmission are peculiar and not commonly shared by other neurotransmitter systems. In particular, GABA-mediated IPSPs exhibit a strong decrement in amplitude following repetitive stimulation of the afferent pathway. Such a phenomenon has been observed in various brain and spinal neurones in vivo and in vitro (Curtis and Eccles, 1960; Ben-Ari et al., 1979; Connors et al., 1982; McCarren and Alger, 1985). Similarly striking is the rapid fading of neuronal responses to exogenously-applied GABA (Krnjevi6, 1981). Even more surprising is the finding that GABA directly depresses GABAergic IPSPs (Nelson et al., 1977; Andersen et al., 1980; Deisz and Prince, 1989). The overall impression given by these data is of a considerable lability of inhibitory processes and such a phenomenon is likely to have important implications for the cellular mechanism generating seizure activity in the brain. For this reason it seems appropriate to comment on some general aspects concerning the regulation of GABAergic transmission. The many factors governing the size (and duration) of IPSPs can be divided into pre- and postsynaptic

56

L. SIVILOTTIand A. NlSTRt

ones. Among the latter it is possible to distinguish energy-dependent pump. It seems probable that the between those pertaining to the GABA receptor CI- gradients and the membrane pump efficiency are complex and those determining the general electro- strongly related to the degree of maturation of the physiological properties of cells. For instance, neurones (Ben-Ari et al., 1989), the ambient temperachanges in the neuronal conductance at rest and ture and the cell metabolic conditions, including in hence in the electrotonic length (i.e. the "cable" particular the extracellular levels of K ÷ which is properties) of a neurone will influence the efficiency co-transported with CI- (Thompson and Gfihwiler, of synaptic transmission in general, including that at 1989b). These factors might explain why different GABAergic synapses. There is, however, no evidence investigations have reported either a substantial that the frequency-dependent depression of GABA- (Huguenard and Alger, 1986; Thompson and Gfihergic transmission is due to these non-specific alter- wiler, 1989a,b,c) or a minimal (Deisz and Prince, ations (Thompson and Gfihwiler, 1989a). It seems 1989; Harrison, 1990) shift in the C1 equilibrium more likely that any postsynaptic change is more potential following repetitive activation of postsynintricately tied to the GABA receptor complex. One aptic GABA receptors. An attenuation (or even obvious possibility is the desensitization of GABA polarity inversion) of the IPSP due to accumulation receptors (Krnjevi6, 1981; Thalmann and Hershkow- of intracellular C1- might reduce the efficiency of itz, 1985): carefully conducted kinetic studies of inhibition because the membrane potential will not be muscimol binding to rat brain membranes have dis- removed by GABA from the threshold value for closed the highly complex nature of the lig- excitation. Nevertheless, since the GABA-activated and-receptor interaction (Yang and Olsen, 1987a). CI- conductance may show outward rectification as The "native" receptor conformation would be con- previously discussed in Section 3.2, this situation will verted by the agonist to a lower affinity state, prob- be mitigated by the fact that the conductance increase ably indicative of desensitization: one should contrast developing during the IPSP will be larger and thus this situation with the increase in affinity observed at capable of "shunting" the postsynaptic membrane the nicotinic cholinergic receptor following desensi- from incoming signals. Even those studies which have tization. While GABA receptor desensitization does detected an important contribution of CI- shifts to occur also under controlled electrophysiological con- IPSP depression have concluded that additional preditions (Thalmann and Hershkowitz, 1985), it is a synaptic mechanisms must be operative under these slowly developing phenomenon which requires time conditions (Thompson and Giihwiler, 1989c). In parperiods ( > 0.5 rain) in excess of those needed to ticular, some experimental observations could be observe the fast onset, frequency-dependent decre- fully accounted for, should GABA reduce the presynment of the IPSP. Another adaptive change in GABA aptic liberation of GABA itself over a rapid time receptor function observed in acutely dissociated and scale. These observations include the depression of internally perfused neurones is a gradual rundown the synaptic conductance during repetitive IPSPs over several minutes. Albeit slower than desensitiza- (Deisz and Prince, 1989) due to a progressive retion, rundown will also drastically reduce the ampli- duction in the transmitter available for its activation, tude of GABA-evoked responses to 10% of their and the IPSP inhibition by exogenous GABA (Deisz original size (Chen et al., 1990). Interestingly, the and Prince, 1989) or by GABA uptake inhibitors, rundown phenomenon is strongly attenuated by which raise the extracellular concentration of GABA adding Mg 2+ and ATP to the cell cytoplasm while it itself (Dingledine and Korn, 1985; Deisz and Prince, is accelerated by raising intracellular Ca 2+ (Gyenes et 1989). Strong evidence to support this view derives al., 1988; Chen et al., 1990; cf. also depression of, from a study on in vitro hippocampal neurones from GABA responses by Ca2+; Inoue et al., 1986). These which inhibitory currents identified as monosynaptic results have led to the hypothesis that the postsyn- were recorded under voltage-clamp (Harrison, 1990). aptic GABAA receptor is maintained in a fully active Furthermore, this notion is consistent with the prestate by a phosphorylation process catalyzed by a synaptic origin for the depression of inhibitory postMgZ+-ATP dependent protein kinase (Chen et al., synaptic responses at the crayfish neuromuscular 1990). Such a system would normally prevent run- junction where GABA is unequivocally recognized down, but would be opposed by the receptor dephos- to be the inhibitory transmitter (Aickin and Deisz, phorylation brought about by an intracellular 1981). phosphatase (perhaps calcineurin) activated by a rise A rather interesting feature of the presynaptic in intracellular free Ca 2+. It is worth mentioning that action of GABA is the identity of the autoreceptor receptor phosphorylation dependent on high levels involved. Baclofen strongly reduces IPSPs (or their of energy substrates has also been demonstrated for underlying currents) by a presynaptic action (Misgeld excitatory amino acid receptors (MacDonald et al., et al., 1984; Deisz and Prince, 1989; Thomson and 1989). G~ihwiler, 1989c; Harrison, 1990) suggestive of a When considering other postsynaptic mechanisms GABA B receptor-mediated phenomenon. As prepromoting the depression of the IPSP, no experimen- viously discussed (Section 4.2), GABA B receptors are known to operate via distinct membrane effector tal support for a use-related, direct block of CIchannels has been found (Thompson and G~ihwiler, systems. The one implicated in the presynaptic regu1989a,b). A distinct possibility is, however, that there lation of GABA release is probably interfering with is, during repetitive activity, a rapid and considerable Ca 2÷ mechanisms (Deisz and Prince, 1989) and, as shift in the CI- gradient across the neuronal mem- such, is little sensitive to antagonism by the receptor brane with consequent reduction in the amplitude of blocker phaclofen or the G protein inhibitor, pertusthe IPSP. In most central neurones C1- is not dis- sis toxin (Harrison, 1990). Neurochemical data tributed passively and is commonly extruded via an have recently demonstrated a presynaptic, GABA-

GABA I~CEPrORMECHANXSMS mediated regulation of GABA release from synaptosomes (prepared from the rat, Bonanno et al., 1989a, or human, Bonanno et al., 1989b, brain) and from rat brain slices (Raiteri et al., 1989a). Although the ability of GABA to inhibit its own release has been reported some time ago (Mitchell and Martin, 1978; Brennan et al., 1981), most subsequent studies have shown that baclofen is more effective in producing such an effect, while muscimol is minimally active (Waldmeier et al., 1988; Bonanno et al., 1989a). Interestingly, the ability of GABA Bligands to depress release is more evident when the frequency of electrical :timulation (Raiteri et al., 1989a) and the extracellular K ÷ levels (Bonanno et al., 1989a) are relatively low. There is also evidence for some regional heterogeneity in the presynaptic GABA receptors. In fact, those present on brain synaptosomes are blocked by phaclofen (Raiteri et al., 1989b; but see different electrophysiological data by Harrison, 1990) while those on spinal cord synaptosomes are insensitive to baclofen, phaclofen or muscimol but still sensitive to GABA. It appears that the release of GABA is not only controlled by GABA itself but also by other neurotransmitters. Activation of adrenoceptors (both of the ~ and ~t2 type) can facilitate such a release (Pittaluga and Raiteri, 1988) and similar results are produced by choline, the natural precursor and metabolite of acetylcholine (Pittaluga and Raiteri, 1987). One might envisage a subtle interplay between various neurotransmitters in enhancing or depressing the degree of inhibitory inputs reaching postsynaptic cells. In conclusion, GABAergic neurotransmission is intrinsically modulated in a complex manner. Even a very simplified scheme can suggest that, following repetitive stimulation of the afferent pathway, there is a postsynaptic decline in the IPSP partly due to a rapid shift in the transmembrane CI- gradient and to slow desensitization of GABA receptors. This is associated with presynaptic autoinhibition (predominantly via GABA Breceptors) produced by extracellular GABA itself. The labile nature of GABAergic transmission might indicate that it is a primary causative factor for the onset of epileptic convulsions (Dichter and Ayala, 1987). The future challenge will be to clarify fully how presynaptic GABA receptors operate and their biochemical nature. Ideally, it would then be possible to design GABA antagonists selective against these receptors and potentially capable of anticonvulsive activity, if their effect were to enhance GABA release. Even if it looks at present a paradox, the concept of inhibiting an autoinhibitory process might prove itself useful for devising future new therapeutic strategies against epilepsy.

6. AN UNUSUAL GABA RECEPTOR IN CENTRAL VISUAL PATHWAYS Electrophysiological recordings of EPSPs elicited by optic nerve axons on neurones of the frog optic tectum in vitro have disclosed that GABA applied by fast superfusion enhances, in a striking fashion, excitatory neurotransmission in this brain area (Nistri and Sivilotti, 1985; Fig. 3). Curiously, this facilitation is

57

equally observed regardless of whether the amplitude of the postsynaptic potentials is maximal or submaximal and, unlike many other GABA-mediated responses, does not show fading even following sustained exposure to this amino acid. Intracellular recordings have confirmed that GABA does not largely increase the input conductance of these cells but substantially lowers their threshold for generating action potentials and impairs the spike accommodation mechanism despite only small changes in membrane potential (Sivilotti, 1988; Nistri and Siviiotti, unpublished). A similar result has also been obtained from the in vitro guinea pig superior colliculus (Arakawa and Okada, 1988), which, in mammals, is the brain area homologous to the optic tectum of the frog. In the case of mammalian neurones the nature of the action of GABA is clearly dose-dependent with inhibition resulting from application of very high concentrations of GABA while an excitatory effect manifests itself following much lower doses. The sustained, excitatory response to GABA displays unusual pharmacological characteristics which suggest the existence of a novel receptor type, distinct from the types A and B previously discussed. In fact, while muscimol and 3-aminopropanesulphonic acids are powerful agonists in this system, bicuculline, even in high concentrations (0.1 mM), is a very weak antagonist (Nistri and Sivilotti, 1985; Sivilotti and Nistri, 1989). Conversely, picrotoxin is a selective blocker of these GABA-induced responses. Neither barbiturates nor benzodiazepines can potentiate the effects of GABA: pentobarbitone, although possessing a weak GABAmimetic action, only antagonizes GABA, while midazolam is completely ineffective against it (Sivilotti and Nistri, 1989). The GABAB agonist baclofen does not mimic the effect of GABA (Sivilotti and Nistri, 1988). Although the pharmacology of the GABA recognition site appears unconventional, these responses mediated by GABA are more typical in their dependence on extracellular CI- (Sivilotti and Nistri, 1989). It appears that the transmembrane CI- gradient of tectal neurones is maintained by the operation of a CI- pump, which is blocked by ammonium ions (Mazda et al., 1990). Furthermore, since the CIchannel blocker penicillin antagonizes responses to GABA as well as to glycine (Mazda et al., 1990), it seems likely that both aminoacids activate a similar CI- channel (as already proposed for other central mammalian neurones; Bormann et al., 1987) although they do so via distinct receptors. Indeed, the effect of glycine on the frog optic rectum is selectively suppressed by strychnine and insensitive to picrotoxin (Sivilotti and Nistri, 1986). All these results indicate that in the optic tectum and superior colliculus there is a GABA receptor system comprising some characteristics of GABA A receptors but lacking many others. Curiously, conventional GABAA receptors are found on the axons of the retinal ganglion ceils which project to and synapse with the tectal neurones (Nistri and Sivilotti, 1988). One might question why typical receptors can be detected on presynaptic fibres but not on the postsynaptic cells. Although this situation may look anomalous, it is perhaps relevant to note that recent studies of molecular biology have shown how

58

L, SIVILOTTI and A. NISTRI

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FIG. 3. Effects of GABA on optic nerve-evoked synaptic responses of the frog optic tectum, a: Records of synaptic events evoked by submaximal or supramaximal stimulation of the optic nerve. U~ and U2 indicate two distinct postsynaptic excitatory potentials. GABA (middle tracings) increases the amplitude of both responses regardless of stimulus intensity. Such a phenomenon is reversible on wash (bottom tracings), b: Dose--response curves for the enhancement of the U~ (top) and U 2(bottom) synaptic responses by GABA. Abscissae: log concentration of GABA; ordinates: % increase over control synaptic event. Reproduced from Nistri and Sivilotti (1985) with permission.

different combinations of the various GABA receptor subunits can be made experimentally to impart distinct pharmacological properties not typically present in the native G A B A receptor. It looks as if those observations based on laboratory recombination studies may actually be validated by the physiological occurrence of such GABA receptor system in the optic tectum. In view of the important role of this brain area in various behavioural responses as well as for coordinating visual activity (Northmore et al., 1988; Dean et al., 1989), it seems likely that a full understanding of the G A B A receptor systems in these regions will help to construct an integrated picture of the neuronal network underlying these phenomena.

7. GABAERGIC SYNAPTIC TRANSMISSION 7. l. HIPPOCAMPUS

7.1.1. G A B A e r g i c pathways

GABAergic transmission is thought to play a very important role in hippocampal inhibitory mechanisms. In the hippocampus, as in other areas, the localization of GABAergic synapses and neurones can be investigated with a variety of immunocytochemical

techniques, which visualize markers for GABAergic transmission, such as the G A B A synthetic enzyme, glutamic acid decarboxylase (GAD) (Ribak et al., 1978; Somogyi et al., 1983a), the GABA inactivating enzyme, GABA-transaminase (GABA-T) (Nagai et al., 1983) or G A B A itself (Storm-Mathisen et al., 1983). The results of these studies indicate that GABAergic markers are present in all hippocampal laminae to a different extent. GABAergic terminals and fibres form a dense plexus around the somata of pyramidal cells in the C A I - C A 3 regions and around granule cells in the fascia dentata (Ribak et al., 1978; Storm-Mathisen et al., 1983; Somogyi et aL, 1983a; Misgeld and Frotscher, 1986; Woodson et aL, 1989). A particularly high density of GABA-positive puncta surrounds CA3 pyramidal cell somata (Woodson et aL, 1989). GABAergic boutons are not confined to the somata of pyramidal and granule cells, but cover their axon hillock and initial segment (Somogyi et al., 1983a; Soriano and Frotscher, 1989) and are present on both basal and apical dendrites (Somogyi et aL, 1983a; Woodson et al., 1989). Woodson et al. (1989) have also observed that in dendritic fields the density of G A B A positive grains is fairly homogeneous in the C A I - C A 3 areas, but not in the fascia dentata, where the outer third of the molecular layer is more intensely labelled. GABAergic boutons terminate also

GABA RECEPTORMECHANISMS on the somata and processes of hippocampal GABAergic interneurones in the dendritic layers of both CA1-CA3 and fascia dentata (Misgeld and Frotscher, 1986; Freund and Antal, 1988; Woodson et al., 1989). GABAergic cells represent 11% of the total population of hippocampal neurones (Woodson et al., 1989): their morphology is quite heterogeneous and includes most types of short axon neurone, including basket, stellate and horizontal cells (Ribak et al., 1978; Seress and Ribak; Nagai et al., 1983; Woodson et al., 1989), some of which may also contain cholecystokinin and/or vasoactive intestinal polypeptide (Sloviter and Nilaver, 1987). Most of the somata of the GABAergic neurones are found outside the pyramidal layers of CA fields and the granule cell layer of fascia dentata and are chiefly located in the CAI-CA3 dendritic regions (Woodson et al., 1989). Note, however, that Schwartzkroin and Mathers (1978) have described a putative GABAergic interneurone in the guinea pig CAI stratum pyramidale. While most GABAergic terminals in the hippocampus are likely to originate from intrinsic, localcircuit neurones, the hippocampal formation also receives GABAergic afferents from the entorhinal cortex via the perforant path (Germroth et al., 1989) and from the septum probably via the fimbria-fornix (K6hler et al., 1984; Freund and Antal, 1988). Indeed, most GABAergic hippocampal interneurones receive input from GABAergic septohippocampal fibres (Freund and Antal, 1988). The presence of GABAergic synapses is reflected by that of GABA receptors. The autoradiographic study by Bowery et al. (1987) has demonstrated the presence of moderate levels of GABAA receptors in all layers of CA1-CA4 regions and dentate gyrus (see also Chan-Palay, 1978). In the rat stratum radiatum, antibodies raised against purified GABAA receptors have revealed a discontinuous distribution of immunoreactivity (Taguchi et al., 1989). Very high levels of flunitrazepam binding were demonstrated in the stratum lacunosum-moleculare of CA3 and in the molecular layer of the dentate gyrus by the autoradiographical study of Unnerstall et al. (1981). The distribution of GABAa sites is less homogeneous, since the density of baclofen binding in the CAI42A4 pyramidal layer and the fascia dentata granular layer is lower than in the dendritic layers of the same regions (Bowery et al., 1987).

59

In the rat and mouse CAI pyramidal cells and in mouse granule cells in vitro spontaneous depolarizing potentials can be observed if the neurone is impaled with microelectrodes filled with a Cl--containing solution (Alger and Nicoll, 1980a; CoUingridge et al., 1984; Biscoe and Duchen, 1985a). Hyperpolarizing spontaneous IPSPs have been recorded from guinea pig CA3 pyramidal cells in vitro with K-acetate-filled microelectrodes by Miles and Wong (1984). These potentials do not appear to be the result of random quantum release from a single terminal, since they occur in clusters, with a non-random interval distribution (Alger and Nicoll, 1980a; Biscoe and Duchen, 1985b) and since their time-to-peak is quite variable (the latter observation suggests that they originate from different areas of the neuronal membrane; Miles and Wong, 1984). Indeed, most, if not all, of the barrage of spontaneous IPSPs impinging on hippocampal cells may be generated by action potential activity in interneurones, since 0.5-1/~M tetrodotoxin (TTX) suppresses spontaneous IPSPs (Alger and Nicoll, 1980a) or greatly reduces their amplitude and frequency (as does 0.2mM Cd2+; Collingridge et al., 1984). The properties of the synaptic current underlying these spontaneous potentials have been investigated by Collingridge et al. (1984), while the ionic selectivity of these synaptic channels has been described by Biscoe and Duchen (1985a) (see also Sections 3.1 and 3.2). The GABAergic nature of the spontaneous IPSPs is demonstrated by their sensitivity to the GABA antagonists bicuculline (1-100/~M; Alger and Nicoll, 1980a; Collingridge et al., 1984) or picrotoxin (100 #M; Miles and Wong, 1984). IPSP duration and amplitude were enhanced by 50-100 #M pentobarbitone (Alger and Nicoll, 1980a; Collingridge et al., 1984). When spontaneous IPSCs were recorded with single electrode voltage-clamp, 100#M pentobarbitone was found to prolong their decay by more than 4-fold (Collingridge et al., 1984). Spontaneous IPSPs can be blocked via a postsynaptic mechanism by 10-50 #M d-tubocurarine (in guinea pig CA3 neurones; Lebeda et al., 1982) or 0.1-1 mM folic acid (in rat CAI neurones; Otis et al., 1985). lontophoresis of the benzodiazepine flurazepam enhanced the amplitude of spontaneous IPSPs in mouse dentate granule cells (Biscoe and Duchen, 1985b). 7.1.2.2. C A I - C A 3 : characteristics o f evoked I P S P s

7.1.2. G A B A e r g i c synaptic potentials

The first indication that GABA could be an important inhibitory transmitter in the hippocampus came from the work of Biscoe and Straughan (1966), who observed GABA-induced depression of spontaneous and glutamate-evoked firing of hippocampal cell in vivo. Subsequent studies have aimed at a clarification of the inhibitory synaptic pathways using GABA as a transmitter. 7.1.2.1. Spontaneous events

The occurrence of spontaneous IPSPs was first described in the rat hippocampal slice by Alger and Nicoll (1980a).

The first intracellular demonstration of evoked inhibitory synaptic potentials in the hippocampus dates back to the work of Kandei and co-authors (1961) on CA2-CA3 pyramidal cells in the cat in vivo. These authors showed that fornix stimulation produced C1--sensitive synaptic potentials. Their results were extended by Andersen et al. (1963a, 1964a,b), who found that CA3 pyramidal cells in the cat in vivo responded with hyperpolarizing synaptic potentials to local stimulation or to the stimulation of a variety of afferent pathways, including commissural, septal and fimbrial inputs. Andersen and co-workers also demonstrated that the resulting hippocampal inhibition was mediated by activation of interneurones synapsing mainly on the somata of pyramidal cells.

60


They suggested that the best candidate for the role of inhibitory interneurone in these circuits is the basket cell. Evidence for the GABAergic nature of fimbrialevoked inhibition was put forward by Curtis et al. (1970b) who found that bicuculline iontophoresis onto C A I - C A 2 neurones in the cat in vivo reduced the inhibition of single unit firing caused by GABA iontophoresis or fimbrial stimulation. It should be noted that, unless chronic deafferentation is carried out (see Kandel et al., 1961), most of the fibre tracts stimulated in the above-mentioned studies are mixed, e.g. contain both afferent and efferent fibres. In more recent work, the use of the hippocampal slice preparation has allowed investigators to stimulate fairly selectively pyramidal cell axons (antidromic stimulation) or afferent fibres that synapse onto the pyramidal cells (orthodromic stimulation) and to compare the evoked potentials. Even so, caution must be exercised in the interpretation of such data, given the possible spread of the electrical stimulus to neighbouring structures. For instance, a "'pure" antidromic response evoked in CA I pyramidal cells by alvear stimulation can be contaminated by an orthodromic component if the stimulation activates adjacent stratum oriens fibres (see Alger, 1984). Conversely, orthodromic stimulation of sufficient intensity will evoke firing in pyramidal cells and consequently the activation of recurrent circuits similar to those involved in the generation of antidromic responses. 7.1.2.3. C A I - C A 3 : antidromic responses Axons originating from hippocampal pyramidal cells form identifiable fibre tracts within the hippocampus such as the alveus (from CA1 neurones) and the Schaffer collaterals (from CA3 neurones). Stimulation of these fibres (or of the chronically deafferented fornix) evokes in the cellular field of origin a monophasic fast hyperpolarizing potential (Fig. 4, bottom), which has a threshold lower than that of the antidromic spike (Kandel et al., 1961; Dingledine and Langmoen, 1980; Biscoe and Duchen, 1985c). This

Control

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fast IPSP is associated with a decrease in the input resistance of the pyramidal cell (Dingledine and Langmoen, 1980) and has a reversal potential in the region of - 65 to - 75 mV in neurones impaled with K methylsulphate or K acetate microelectrodes (Andersen et al., 1980; Alger and Nicoll, 1982b; Biscoe and Duchen, 1985c). The IPSP appears to be due to an increase in the membrane permeability to CI , since its amplitude decreases after replacement of part of the C1- in the extracellular medium with the impermeant anion is ethionate (Alger and Nicoll, 1982b). These results are consistent with the observation by Kandel and co-authors (1961) that intracellular CI- injection reverses the antidromic IPSP. The involvement of G A B A A receptors in the production of these synaptic potentials is proved by their blockade by iontophoresis of bicuculline methiodide (Alger and Nicoll, 1982b) or by superfusion with 1-100/IM picrotoxin (Alger, 1984; Miles and Wong, 1984). When the fast IPSP is suppressed by picrotoxin, antidromic stimulation elicits a biphasic response in rat CAI cells, constituted by an EPSP-slow IPSP sequence (Alger, 1984). The failure to observe a slow picrotoxin-insensitive IPSP after a lesion of the stratum oriens argues for the generation of such biphasic responses via stimulus spread to orthodromic afterents in this layer (Newberry and Nicoll, 1984b). Nevertheless, it has been shown by Miles and Wong (1984) in the guinea pig hippocampus that stimulation of a pure axonal projection from CA3 to CA! stratum oriens evokes in CA3 pyramidal cells a fast IPSP which is converted by 100 # i picrotoxin to an EPSP-slow IPSP sequence. Given the anatomical separation of this fibre tract from orthodromic afterents to the CA3 region, Miles and Wong (1984) have suggested the possibility of recurrent excitatory loops for CA3 cells (see also MacVicar and Dudek, 1980). Proctor et al. (1986) have observed a biphasic hyperpolarizing response of rat CA1 neurones to alveus stimulation. The antidromic generation of this potential is supported by the failure of pentobarbitone to reverse the fast IPSP into a depolarizing potential (see below).

Recovery

Or~

400 ms Anti

FIG. 4. Differential antagonism by bicuculline of orthodromic and antidromic IPSPs in a rat hippocampal neurone in vitro. Top records: responses evoked by orthodromic stimuli show fast IPSP (left) which is blocked by applying bicuculline methiodide to the dendritic region (note inset showing double action potential resulting from block of inhibition). The late IPSP is not affected by bicuculline (compare superimposed traces in right panel). Bottom records: IPSPs induced by antidromic stimulation are little sensitive to dendritic application of bicuculline. Reproduced with permission from Alger and Nicoll (1982b).

GABA RECEPTORMECHANISMS A somatic location of the synapses generating the fast antidromic IPSP (as originally suggested by Andersen et al. (1964a) on the basis of a marked positivity in the extracellular field potential recorded from stratum pyramidale) is supported by the sensitivity of this response to somatic iontophoresis of bicuculline methiodide, TTX or Cd 2÷ (Alger and Nicoll, 1982b; Newberry and Nicoll, 1984b) and its relative lack of sensitivity to dendritic application of bicuculline (Fig. 4) (Alger and Nicoll, 1982a). Superfusion with 100-125 #M pentobarbitone (or 5-10 fold higher concentrations of phenobarbitone) results in a dramatic enhancement of the amplitude and duration of the IPSP (Alger and Nicoll, 1982b; Proctor et al., 1986). This effect is reproduced by 10/tM (+)etomidate (Proctor et al., 1986). The marked potentiation of hippocampal IPSPs by barbiturates was first observed for responses evoked by local stimulation in CAI neurones by Nicoll et al. (1975), who compared nitrous oxide- with pentobarbitone-anaesthetized animals. Half-decay time was strikingly prolonged (2.5-5-fold) in cats anaesthetized with pentobarbitone or in cats anaesthetized with nitrous oxide who were given 10 mg/kg pentobarbitone i.v. If orthodromic contamination of the antidromic response is excluded by a transverse cut of the stratum oriens, the polarity and reversal potential of the fast IPSP are unaffected by pentobarbitone (Alger and Nicoll, 1982b). The amplitude of antidromic IPSPs is enhanced by 0.1-1/~M diazepam only for submaximally evoked responses; higher diazepam concentrations (10#M) have a general depressant effect (Alger and Nicoli, 1982b). The antidromic IPSP is generated by a feed-back circuit which is formed by recurrent collaterals of the efferent axons of pyramidal cells synapsing onto inhibitory interneurones. This mechanism is supported by recordings from pairs of cells in the hippocampus (MacVicar and Dudek, 1980; Knowles and Schwartzkroin, 1981; Miles and Wong, 1984). Some interneurones can be identified as such on the basis of their intrinsic electrophysiological properties (high frequency of firing, spontaneous action potentials, pronounced afterhyperpolarization; Knowles and Schwartzkroin, 1981), apart from the location of their somata (see Section 7.1.1). Knowles and Schwartzkroin have observed that CAI pyramidal cells consistently excite interneurones, whereas interneurones inhibit pyramidal cells: in both cases the short latency of the synaptic potentials generated in the postsynaptic cell argues for a monosynaptic connection of the pairs. When both impaled cells are pyramidal neurones, activation of one elicits a disynaptic IPSP in the other (Knowles and Schwartzkroin, 1981; see however MacVicar and Dudek, 1980, for excitatory interactions between pyramidal cell pairs). This interaction can be fairly strong: Miles and Wong (1984) have recorded from a pair of cells in which 20% of action potentials in the presynaptic element were followed by a disynaptic IPSP in the postsynaptic neurone. Recurrent inhibition can be autogenic: indeed, in 15% of CA3 neurones spontaneous action potentials are followed within 15 ms by an IPSP (Miles and Wong, 1984).

61

While GABAergic synaptic contacts onto pyramidal cells somata may be provided mostly by basket cells, it is difficult to ascribe with any degree of certainty the mediation of the recurrent IPSP to a single subset of interneurones. It would seem likely that axo-axonic GABAergic cells synapsing onto the axon initial segment may also contribute to feed-back IPSPs. In fact, because of the electrotonic structure of pyramidal neurones, IPSPs generated at the soma cannot be distinguished by electrophysiological techniques from IPSPs generated at the axon initial segment. Furthermore, recurrent IPSPs can be demonstrated in hippocampal cells of the newborn kitten at a stage when basket cell axosomatic synapses are absent and axoaxonic synapses are already found (Somogyi et al., 1983a). Another candidate may be the interneurone described in stratum pyramidale by Schwartzkroin and co-workers (Schwartzkroin and Mathers, 1978; Knowles and Schwartzkroin, 1981). It is worth mentioning that, on the basis of the relative sizes of the IPSP evoked by fibre tract stimulation vs the "unitary" monosynaptic IPSP evoked by single neurone stimulation, Miles and Wong (1984) have suggested that each pyramidal cell may receive synaptic contacts by up to 15 interneurones. The accuracy of such an estimate obviously depends on how representative the described unitary IPSPs sample is of the "population" of IPSPs that contribute to the synaptic potential evoked by electrical stimulation. A bias in the recorded pairs of cells towards over-representation of somatic synapses would lead to a serious underestimate of interneurone convergence onto hippocampal pyramidal cells. 7.1.2.4. C A I - C A 3 : orthodromic responses Electrical stimulation of orthodromic inputs elicits in hippocampal pyramidal neurones a synaptic response which is much more complex than the fast recurrent IPSP produced antidromically. Characteristically, the orthodromic response consists of an EPSP (which may cause the cell to fire) followed by a hyperpolarizing IPSP: Fig. 4 shows that if the intensity of stimulation is sufficient, the IPSP is clearly biphasic and comprises a fast and slow component (Alger and Nicoll, 1982b; Newberry and Nicoll, 1984b; Alger, 1984; Knowles et al., 1984; Biscoe and Duchen, 1985c). In addition to the synaptic potentials, an even slower hyperpolarization can be observed when the EPSP has produced an action potential. This component is probably due to activation of a CaZ+-depen dent K + conductance (Alger and Nicoll, 1980b; Hotson and Prince, 1980; Newberry and Nicoll, 1984b). The properties of the fast IPSP are in part similar to those of the antidromic IPSP and accord with those expected of a GABAA-generated synaptic potential. Thus, the fast IPSP is associated with a very pronounced decrease in the input resistance of the pyramidal cell (Knowles et al., 1984). This phenomenon appears to be due to an increase in CI- conductance. As shown in Fig. 5, the fast IPSP reversal potential, normally 10-20mV more negative than the resting membrane potential (Ben-Ari et al., 1981;

62

L. SIVILOTTIand A. NISTRI Amplitude of i.p.s.p. (mY) 8

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-90 Membrane potential ImVI FIG. 5. Effect of changing membrane potential on fast and slow IPSPs of rat hippocampal neurone in vitro. A: Traces of orthodromically-induced fast and slow IPSPs (their respective peaks are marked by arrows at - 64 mV). Note different reversal potential values. B: Plot of IPSP amplitudes (open circles: fast IPSP; filled circles: slow IPSP) against membrane potential. Compare dissimilar reversal level for fast (at -71 mV) and for slow (at -85 mV) IPSP. Note also strong rectification of slow IPSP amplitude which declines as the cell membrane is depolarized. Reproduced with permission from Newberry and Nicoll (1985).

Knowles et al., 1984; Biscoe and Duchen, 1985c; Misgeld et al., 1986), becomes positive to the resting membrane potential following intracellular CI- injection, as shown in the cat in vit, o by Eccles et al. (1977) (note that IPSPs were evoked by local stimulation of the hippocampal surface), in the rat in vivo by Ben-Ari et al. (1981) (IPSPs were evoked by fimbrial or entorhinal stimulation) and in hippocampal slices by Newberry and Nicoll (1984b), Biscoe and Duchen (1985a) and Misgeld et al. (1986). Accordingly, substitution of CI- with isethionate or other impermeant anions in the extracellular medium of in vivo preparations produces a depolarizing shift in the fast IPSP reversal potential (Alger and Nicoll, 1982b; Knowles et al., 1984). The involvement of GABAA receptors in the generation of the fast IPSP is supported by its sensitivity to the GABAA antagonists, picrotoxin (bath-applied at 1-10pM; Knowles et al., 1984) or bicuculline added to the superfusing medium at 1-10/~M (Knowles et al., 1984) or iontophoresed as bicuculline methiodide (Alger and Nicoll, 1982b). This conclusion is reinforced by the identity of the reversal potentials for GABA and the fast IPSP (Andersen et al., 1980; Ben-Ari et al., 1981; Misgeld et al., 1986). Particular caution should be exercised in the interpretation of data on the apparent reversal potential of the orthodromic fast IPSP because of the probably composite nature of this signal. In fact, even prescinding from its possible overlap with the EPSP and the late IPSP, the fast IPSP appears to be generated mainly on the dendrites of the pyramidal cells, as demonstrated by its sensitivity to dendritic iontophoresis of bicucuiline methiodide (Fig. 4)

(Alger and Nicoll, 1982b). This synaptic potential may therefore be produced at a location electrotonically distant from the recording electrode and poorly controlled by intracellularly applied currents used to reverse the polarity of the potential. Furthermore, depending on the proportion of pyramidal cells that reach firing threshold at any given intensity of orthodromic stimulation, the orthodromic fast IPSP may comprise a variable somatic component elicited via recurrent collateral fibres. Note also that Alger and Nicoli (1982b) did not exclude that the fast IPSP may also be generated by inhibitory synapses located on the pyramidal cell soma and activated by orthodromic stimulation. While diazepam (0.1-1/aM) enhances submaximal IPSPs, barbiturates have a complex effect on the orthodromic fast IPSP (Alger and Nicoll, 1982b). Bath-applications of 100/~ M pentobarbitone (or 5-10 times higher concentrations of phenobarbitone) transform the hyperpolarizing fast IPSP into a biphasic hyperpolarizing-depolarizing response. The depolarizing component of the IPSP in the presence of barbiturates is strongly enhanced by GABA uptake inhibitors or by recording at 22°C rather than 37°C. This response is generated in the dendrites by GABAA receptor activation, since it is abolished by dendritic iontophoresis of bicuculline methiodide or TTX (Alger and NicoU, 1982b). These authors (1982a) have hypothesized that barbiturates may sensitize extrasynaptic, depolarizing GABAA receptor mechanisms (distinct from the conventional synaptic, hyperpolarizing GABAA receptors) to synapticallyreleased GABA. Under normal conditions, depolarizing IPSPs would not be detected because uptake

GABA RECEPTORMECHANISMS limits the diffusion of GABA from the synapses. Such conclusions are supported by the observation of depolarizing bicuculline-sensitive synaptic responses in hippocampal slices treated with 4-aminopyridine, which can be expected to increase synaptic transmitter release (Avoli and Perreault, 1987). A late depolarizing potential following mossy fibre stimulation has also been described by Thalmann (1988) in CA3 pyramidal cells after blockade of the late IPSP by intracellular GTP-7-S or by in vivo pretreatment with pertussis toxin (see below). This depolarizing potential was enhanced by pentobarbitone. The ionic mechanisms underlying depolarizing IPSPs have not been established with certainty and could involve either the activation of a mixed Ci-/cation conductance or the activation of a CI conductance in parts of the dendritic tree having an outward electrochemical gradient for CI . Generation of fast IPSPs also occurs on basal dendrites in response to stimulation of stratum oriens afferent fibres: the resulting potential is mainly depolarizing in the presence of pentobarbitone (Alger and Nicoll, 1982b). The properties of the slow IPSP are quite distinct from those of the fast orthodromic IPSP or the recurrent IPSP (Fig. 5). The slow IPSP appears only at higher intensities of stimulation (Newberry and Nicoll, 1984b), has a much slower time course than the fast IPSP, lasting hundreds of ms (Newberry and Nicoll, 1984b; Alger, 1984; Hablitz and Thalmann, 1987), and is associated with a relatively modest conductance increase (Knowles et al., 1984; Alger, 1984). Determining the reversal potential for the slow IPSP can be difficult, since membrane rectification opposes the hyperpolarizing effect of steady current injections into pyramidal cells (Knowles et al., 1984; Alger, 1984). More reliable estimates of the slow IPSP reversal potential can be obtained by blocking inward rectifiers by the addition of I mu Cs ÷ to the extracellular medium. The resulting value of - 9 5 mV is consistent with the activation of a K ÷ conductance during the slow IPSP (Hablitz and Thalmann, 1987). Voltage-clamp experiments confirm this conclusion, showing that the reversal potential for the late IPSC ( - 9 9 mV) becomes less negative when extraceilular K + is raises (Hablitz and Thalmann, 1987; for the voltage-dependence characteristics of the slow IPSC, see Section 4.2). Similar results were obtained by Alger (1984) in current-clamp experiments in the presence of raised extracellular K ÷ concentrations to make reversal of the slow IPSP easier. Changes in extra- or intracellular CI- levels have little (Knowles et al., 1984) or no effect on the slow IPSP (Newberry and Nicoll, 1984b; Biscoe and Duchen, 1985c). The slow IPSP is not blocked by GABAA antagonists: indeed, bath-applied bicuculline methiodide enhances the slow IPSP, possibly by removing GABAA-mediated inhibition of GABAergic interneurones (Newberry and Nicoll, 1984b). The slow IPSP is sensitive to GABAa receptor antagonists such as phaclofen (see Fig. 2): this compound (0.2~).5mM) blocks the slow IPSP evoked in rat CAI pyramidal cells by high intensity stimulation of the stratum radiatum, leaving the fast IPSP (observable in isolation at lower stimulation intensities) unchanged (Dutar and Nicoll, 1988a: Soltesz et al., 1988). A similar effect is JPN 3&I--E

63

produced more potently by 2-hydroxysaclofen (50-200/~M; Lambert et al., 1989). Blockers of the coupling of GABAB receptors to K ÷ channels are also effective in suppressing the slow IPSP. For instance, the GABAB slow IPSP is abolished by in vivo pretreatment with pertussis toxin (Dutar and Nicoll, 1988b; Thalmann, 1988) or by intracellular injection of GTP-y-S (Thalmann, 1988). The generation of this response by synaptic mechanisms is supported by its sensitivity to the Ca2+-blocking effects of Cd 2÷ (20/~M; Newberry and Nicoll, 1984b) or Mg 2+ (5 mM; Alger, 1984). Indeed, the slow IPSP was more sensitive than the EPSP to high Mg 2÷, indicating a di- or polysynaptic origin (Alger, 1984). A further proof that the slow IPSP is not produced by the activation of intrinsic voltage- or ion-gated currents is that its amplitude is not correlated to that of the EPSP (Alger, 1984) and is not affected by forskolin, 8-bromo-cAMP or intracellular EGTA, which are blockers of Ca2+-dependent K ÷ conductances (Alger, 1984; Newberry and Nicoll, 1984b; Knowles et al., 1984; Hablitz and Thalmann, 1987). Newberry and Nicoll (1984b) have shown that the slow IPSP is generated at a predominantly dendritic location, since it is spared by somatic iontophoresis of Cd 2÷ or TTX. In conclusion, the GABAergic inhibitory pathways impinging on pyramidal neurones can be summarized as follows. The orthodromic IPSP is mediated by the direct activation of afferent fibres of GABAergic interneurones, synapsing mainly onto the dendrites of pyramidal cells (feed-forward inhibition; Alger and Nicoll, 1982b). Additionally, a somatic inhibitory component may be present either as a direct result of feed-forward mechanisms or because of stimulation of feed-back circuits brought about by activation of the pyramidal cells. As for recurrent inhibition, it is difficult to ascribe feed-forward IPSPs to the activity of a single set of interneurones (say in stratum radiatum or oriens). As Buzs~iki (1984) pointed out in his review, basket cells in the pyramidal layer extend their dendritic arborizations quite far and could receive excitatory synaptic terminals directly from afferent fibres. A possible wiring diagram for the generation of IPSPs by ortho- and antidromic input activation has been put forward by Alger and Nicoll (1982b; see also the discussion of feed-forward hippocampal circuits by Buzs~iki, 1984). 7.1.2.5. Granule cells Comparatively less and more discordant evidence is available on inhibitory synaptic responses recorded from hippocampal granule cells. These responses include both a fast and a slow IPSP, mediated by GABA A and GABAB receptors, respectively. A fast, Cl--dependent postsynaptic potential has been observed following both ortho- and antidromic stimulation in the guinea pig hippocampal slice (Thalmann and Ayala, 1982; Misgeld et al., 1986). This postsynaptic potential has been described as hyperpolarizing by Thalmann and Ayala (1982) and depolarizing by Misgeld et al. (1986), regardless of its ortho- or antidromic generation. Using K acetate- and sulphate-filled microelectrodes, these authors have determined reversal potentials for the GABAA

64

L. SIVILOTTIand A. NISTR1

postsynaptic potential of - 75 and - 62 mV, respectively. This response is, however, likely to be contaminated to an unknown extent by the slow IPSP (see below) and, at least for orthodromic potentials, by the EPSP. Hence, more extensive studies are required to settle this issue, particularly now that blockers of GABAB responses are available. The fast IPSP is blocked by 50/~M picrotoxin (Thalmann and Ayala, 1982) and by a variety of antagonists of GABA A responses, such as penicillin, bicuculline and pentylenetetrazol (Fricke and Prince, 1984), whereas it is not affected by strychnine (i.v. in the rabbit in vivo; Andersen et al., 1966). Some authors (see Dudek et al., 1976) have found that IPSPs can be elicited in granule cells only occasionally. Andersen et al. (1966) have suggested a somatic location of the GABAergic synapses mediating the IPSP on the basis of the depth at which a positive-going waveform in the extracellular field potential (presumably corresponding to the IPSP) reaches its maximum. Nevertheless, this conclusion relies on the assumption that in the unimpaled granule cell the IPSP is hyperpolarizing, and hence a field potential "source" is found at the level of the active synapse. As for the slow IPSP, it has been observed in response to both anti- and orthodromic granule cell stimulation (Thalmann and Ayala, 1982). This potential was sensitive to changes in the extracellular levels of K +, but not CI-, had a reversal potential of - 8 0 to - 9 0 m V and was not blocked by picrotoxin (Thalmann and Ayala, 1982; Biscoe and Duchen, 1985c). Andersen et al. (1966) hypothesized a major role for dentate basket cells in granule cell inhibition, mainly because orthodromic inhibition has pronounced divergence, e.g. is widespread even when stimulation intensity is low and few granule cells are firing. Assuming a recurrent mechanism for IPSP generation, the anatomical correlate for this phenomenon must be a GABAergic interneurone which synapses onto a large number of granule cells. It is, however, likely that both feed-forward and feed-back circuits have a role in granule cell inhibition (for a discussion see Buzs~ki, 1984). Furthermore, there are other interneuronal types which may mediate the inhibition elicited by perforant path stimulation: for instance, Soriano and Frotscher (1989) have described a GABAergic axo-axonic interneurone in fascia dentata. The dendrites of this cell type are mainly in the molecular layer, where perforant path fibres terminate, while its axon synapses on the axon initial segment of granule cells. 7.1.2.6. Interneurones Intracellular data on synaptic responses in interneurones are limited because of the technical difficulty in obtaining intracellular recordings from small cells. Schwartzkroin and Matbers (1978) have obtained recordings from a CAI interneurone type distinct from basket cells: inhibitory synaptic potentials were observed only in response to stratum radiatum stimulation, as EPSP-IPSP sequences. As a result of the long duration of the hyperpolarization, which was associated with a small conductance

increase, it seems likely that a slow IPSP was present. Misgeld and Frotscher (1986) have investigated the electrophysiology of interneurones of the deep hilar region of fascia dentata and stratum lacunosummoleculare of CA3. The interneurones were identified as such on the basis of their morphology as revealed by Lucifer Yellow intracellular staining. Prominent hyperpolarizing IPSPs, blocked by 25 #M bicuculline, were observed in both areas following stimulation of perforant path and local afferent fibres, respectively. In hilar interneurones an IPSP-EPSP sequence was elicited by perforant path afferents: the latency of the initial IPSP was similar to that of the initial EPSP in granule cell neurones in the same slice. This GABAergic control of GABAergic neurones will result in disinhibition in the principal (e.g. pyramidal and granule) cells. 7.2. OLFACTORY CORTEX AND NEOCORTEX

7.2.1. G A B A e r g i c pathways The results obtained with the different histological techniques for the visualization of GABAergic structures agree in showing high densities of GABAergic terminals in these areas. In the olfactory cortex, large numbers of boutons labelled for G A D were found in layer II, surrounding pyramidal cell somata and, to a lesser extent, in layer Ia, on distal apical dendrites of pyramidal cells (Haberty et al., 1987). Extensive investigations into the distribution of GABAergic structures have been carried out in visual areas of the neocortex, e.g. the striate cortex. While in area 17 of the rat GAD-positive boutons are homogenously distributed throughout the cortical layers (Ribak, 1978), in the rhesus monkey a complex pattern of lamination can be observed (Hendrickson et al., 1981). Not only are GABAergic terminals more abundant in some layers (IVa, IVc, the superficial half of layer I and, to a lesser degree, layers II, III and VI) than in others, but they can also be seen to form clusters within layers when the cortex is sectioned on a plane parallel to its surface. Thus, G A D reactivity in layers II and III is concentrated in "dots" which correspond to the areas rich in cytochrome oxidase. These "dots" run in parallel to the centres of the layer IVc ocular dominance columns (Hendrickson et al., 1981). For the neocortex in general, Nagai et aL (1983) have reported that, in the rat, neuropil staining for newly-synthesized GABA-T is highest in layers III and V. GABAergic boutons appear to synapse onto the somata of practically every neocortical neurone (Ribak, 1978): GABA-positive puncta almost completely outline non-GABAergic pyramidal cell perikarya in layers III and V (Ottersen and Storm-Mathisen, 1984). Indeed, more than 90% of the boutons surrounding the pyramidal somata in layers II-III of the cat visual cortex are GAD-immunoreactive (Freund et al., 1983). GABAergic synapses have also been demonstrated on the dendritic shafts of pyramidal neurones and, less frequently, on their dendritic spines and axon initial segments (Ribak, 1978). Synapses on the initial segment of the pyramidal cell axons are almost exclusively GABAergic (Freund et al., 1983). GABAergic

GABA RECEPTORMECHANISMS terminals have also been shown to synapse onto GABAergic cortical neurones (Hendrickson et al., 1981; Somogyi et al., 1983b; Ottersen and StormMathisen, 1984). Both in the olfactory cortex and in the neocortex, neurones that use GABA as their transmitter belong to non-pyramidal cell types. GABAergic cells are found throughout the depth of the olfactory cortex and include, in the opossum, large horizontal cells in layer Ia, small globular cells in Ib and II and multipolar and fusiform cells in the deeper parts of layer III (Haberly et al., 1987). In the neocortex it has been estimated that most non-pyramidal cells are GABAergic, with the notable exception of the small spiny neurones of layer IV (Jones and Hendry, 1986). Ribak (1978) described GAD-immunoreactive cells in the rat visual cortex as having the morphology of stellate neurones, with few or no dendritic spines. These cells are homogeneously distributed throughout the cortical layers (Ribak, 1978; Ottersen and Storm-Mathisen, 1984) and include a variety of morphologically-distinct cell types (Somogyi et al., 1983b), such as basket and axo-axonic or chandelier cells (Freund et al., 1983; Wise, 1985; Jones and Hendry, 1986). Approximately 10% of GABAergic neurones also contain neuropeptides like cholecystokinin, substance P, somatostatin and neuropeptide Y (Hendry et al., 1984; Jones and Hendry, 1986). Nearly all neuropeptide-containing cortical interneurones are also positive for GAD: neurones containing cholecystokinin and GAD may synapse preferentially on the proximal dendrites of pyramidal neurones, whereas the terminals of neurones containing somatostatin, neuropeptide Y and GAD appear to synapse on the distal dendrites and dendritic spines of pyramidal cells (Jones and Hendry, 1986). No peptide reactivity can be demonstrated in basket or chandelier GABA interneurones (Wise, 1985; Jones and Hendry, 1986). Most of cortical GABA derives from intrinsic neurones, as shown by the observation that GABA levels in a cortical slab do not change when the connections between the cortical slab and other areas of the CNS are severed by undercutting (Emson and Lindvall, 1979). There is, however, evidence for the presence of a few GABA-positive fibres in the cortical white matter and in the corpus callosum (Ottersen and Storm-Mathisen, 1984) and for the existence of a GABAergic projection from the tuberal, caudal and postmammillary caudal magnocellular nuclei of the hypothalamus to the neocortex (Vincent et al., 1983). Very high levels of GABA A and GABAB binding sites are present in the frontal cortex and other neocortical areas (Bowery et al., 1987). The distribution of muscimol binding sites has a laminar pattern (Chan-Palay, 1978). Similarly, GABAA-receptor-like immunoreactivity is found in layers Ill-IV of rat neocortex (Taguchi et al., 1989). Lamina IV has the highest level of flunitrazepam binding sites found in the rat brain (Unnerstall et al., 1981). 7.2.2. GABAergic

synaptic potentials neurones

o f principal

The first description of an inhibitory effect of GABA iontophoresed onto neocortical neurones

65

dates back to the work of Krnjevi6 and Phillis (1963) in the cat in vivo. 7.2.2.1. Spontaneous events In the mammalian cortex in vitro no spontaneous IPSPs can be observed at resting membrane potential if recording is carried out with a microelectrode filled with an impermeant anion such as acetate (Connors et al., 1982). Hyperpolarizing spontaneous synaptic potentials can be observed only in approximately 10% of guinea-pig neocortical neurones depolarized by 20 mV from their resting membrane potential (Connors et al., 1982). Depolarizing spontaneous potentials appear when the recording electrode contains C1- ions, both in the olfactory cortex and in the neocortex (Satou et al., 1982b; Galvan et aL, 1985; Tseng and Haberly, 1988). These spontaneous potentials are GABAergic IPSPs, since they are reversibly blocked by 100#M bicuculline methiodide and enhanced and prolonged by 100pM pentobarbitone (Galvan et al., 1985). As in the hippocampus, interneuronal firing plays a role in the genesis of these events, since the spontaneous IPSPs are abolished by application of 1--4/./M TTX (Galvan et al., 1985). 7.2.2.2. Evoked synaptic potentials Early in vivo intracellular studies reported that pyramidal (Betz) cells in the motor cortex can be inhibited by electrical stimulation of a variety of inputs, including the pyramidal tract (Phillips, 1956), specific thalamic nuclei, the cortical surface itself (Branch and Martin, 1958) and the corpus callosum, or by noxious pressure applied to a limb (Krnjevi6 et al., 1964, 1966a). This inhibition is manifested as suppression of spontaneous firing activity (Branch and Martin, 1958) and is reflected by a hyperpolarization of the Betz cell (Phillips, 1956), which may last hundreds of milliseconds (Stefanis and Jasper, 1964). The hyperpolarization is associated with a marked decrease in the input resistance of the neurone (Pollen and Lux, 1966). The inhibition evoked by local cortical stimulation is widely spread through the cortex (Krnjevi6 et al., 1966a) and persists after cortical undercutting, suggesting involvement of intrinsic neurones in its generation (Krnjevi6 et al., 1966b). Field potential studies point to layers II and IV for the localization of the synapses activated by cortical stimulation (Krnjevi6 et al., 1966b): note, however, that this conclusion is based on the premise that the IPSP produced by these synapses is hyperpolarizing in the unimpaled cell, e.g. transmitter release generates a current "source" in the membrane under the synapse. Clear evidence for the GABAergic nature of the IPSP evoked by cortical stimulation was first presented by Krnjevi6 and Schwartz (1967), who showed that this synaptic potential had the same reversal potential as the neuronal responses to iontophoretic GABA and was 10-20 mV negative to the resting membrane potential. Both the IPSP and the GABA reversal potential are shifted to a new value positive to the cell membrane potential by intracellular CI- injection (Krnjevi~ and Schwartz, 1967). Dreifuss et al. (1969) showed that the reversal potential for the IPSP and the GABA response was shifted

66


to the depolarizing direction by prolonged GABA applications. The IPSP evoked in Betz cells by antidromic stimulation of the pyramidal tract has very similar properties to that evoked by local stimulation, despite a shorter duration (100-150ms vs 200400ms) and a more modest conductance increase (Renaud et aL, 1974). In the cat olfactory cortex Biedenbach and Stevens (1969) described an EPSP-IPSP sequence evoked in pyramidal neurones by stimulation of their orthodromic input, the lateral olfactory tract (note, however, that antidromic spikes can sometimes be observed in response to lateral olfactory tract shocks; Satou et al., 1982a). The properties of these synaptic responses have been investigated in depth by Satou et al. (1982a) in the rabbit in vivo. They found that the hyperpolarizing IPSP elicited by lateral olfactory tract stimulation was biphasic, comprising a fast and a slow component, which, as in the hippocampus, manifested the characteristics expected of synaptic potentials mediated by GABAA and G A B A B receptors, respectively. Thus both IPSPs are graded and potentials do not depend upon the previous occurrence of action potentials (Satou et al., 1982a). The fast IPSP is associated with a very pronounced decrease in the cell input resistance and is C1--dependent, since it becomes depolarizing upon intracellular injection of C1- (Biedenbach and Stevens, 1969; Satou et al., 1982a). The slow IPSP is associated with a smaller increase in neurone conductance; its reversal potential is considerably more negative than that of the fast IPSP and is unaffected by intracellular C1levels. While the amplitude of the fast IPSP changes linearly with membrane potential changes, the slow IPSP displays a non-linear behaviour, with decreases in both its amplitude and its associated conductance increase upon displacement of the membrane potential in either the hyperpolarizing or the depolarizing direction (Satou et al., 1982a). Fast IPSPs can also be elicited in the principal cells of the rabbit olfactory cortex by stimulating the olfactory bulb, the anterior commissure or the deeplying structures of the olfactory cortex (Satou et al., 1983a). A pharmacological validation of the role of G A B A as the principal mediator of cortical inhibition is provided by the finding that i.v. bicuculline antagonizes the inhibition of cat pericruciate cortical neurones following local or pyramidal tract stimulation (Curtis et al., 1970b). Curtis and Felix (1971) have strengthened this conclusion by demonstrating that bicuculline iontophoresis reduces the inhibitory effect of both iontophoretic G A B A and local or pyramidal tract stimulation. It should be noted that synaptically-evoked inhibition is always less sensitive to bicuculline antagonism than iontophoretic GABA responses and that the cortical inhibition elicited by the stimulation of the nucleus ventralis posteromedialis in the thalamus is virtually unaffected by bicuculline. The introduction of in vitro slice techniques has made it possible to obtain more stable intracellular recordings. On the other hand, since pyramidal cell axons and cortical afferent fibres intermingle in the subcortical white matter, it is impossible to stimulate orthodromic or antidromic pathways selectively in the neocortical slice preparation. Stimulation at mod-

crate intensity of either the white matter or the pial surface of a neocortical slice elicits a depolarizing synaptic potential in the pyramidal cells, while higher stimulation intensities produce biphasic depolarizing-hyperpolarizing responses (Connors et al., 1982). The complex origin of these evoked synaptic potentials is revealed by depolarizing the neurone with current injection: the early depolarizing phase becomes a depolarizing-hyperpolarizing potential, which is followed (for sufficiently high stimulus intensities) by a more negative slow hyperpolarization. The response is thus constituted by an EPSP followed by a fast IPSP (depolarizing at resting membrane potential) and a slow IPSP (Connors et al., 1982; McCormick et al., 1985; Avoli, 1986; Howe et al., 1987a). Similar results were obtained in the olfactory cortex slice with lateral olfactory tract stimulation (Scholfield, 1978a; Tseng and Haberly, 1988). In both areas, the fast IPSP is associated with a pronounced conductance increase (Scholfield, 1978a; Connors et al., 1982; Avoli, 1986; Howe et al., 1987a; Connors et al., 1988; Tseng and Haberly, 1988). Accurate measurements of the fast IPSP reversal potential are made difficult by its overlapping with the EPSP (Weiss and Hablitz, 1984; Tseng and Haberly, 1988). Available estimates range from approximately - 6 0 to - 6 5 mV in the olfactory cortex (Scholfield, 1978a; Tseng and Haberly, 1988) to - 7 0 or - 8 0 mV in the neocortex (Howe et al., 1987b; Connors et al., 1988; but cf. the value of - 5 7 to - 6 5 mV reported by Weiss and Hablitz, 1984): these values overlap (or are depolarized to) the resting membrane potential of pyramidal cells in vitro (Connors et al., 1982). The opposite polarity of the fast IPSP observed in vivo could be due to the lower, more depolarized cell membrane potentials reported for such preparations, which in turn could be due to impalement damage to cells or to the presence of a tonic barrage of depolarizing potentials impinging onto the pyramidal neurone in the intact animal. Differences in the equilibrium potential values for the ion that mediates the fast IPSP, namely C1 , are also possible and might be attributed to reduced activity of C1 membrane pumps in vitro. Nevertheless, cortical slices are often maintained at temperatures near physiological values (35-37 ° by Connors et al., 1982, and McCormick et al., 1985; 36-37°C by Howe et al., 1987a,b) which should ensure the adequate functioning of CI- pumps. Note, however, that the fast IPSP reversal potential appears to be more depolarized whenever olfactory cortex slices (Scholfield, 1978a; Tseng and Haberly, 1988) or neocortical slices (Weiss and Hablitz, 1984) were maintained at lower temperatures. It must also be kept in mind that the degree of temporal overlap between the EPSP and the fast IPSP also depends in a complex way upon ambient temperature. The reversal potential of the fast IPSP is shifted in the depolarizing direction by rises in intracellular CIconcentration (Scholfield, 1978a; Avoli, 1986; Tseng and Haberly, 1988), but is comparatively insensitive to changes in the extracellular K + levels (Avoli, 1986; Howe et al., 1987a), a somewhat surprising finding if one considers that the concentration of extracellular K + should influence the transmembrane transport of Cl-.

GABA RECEPTORMECHANISMS The fast IPSP shows the pharmacological properties of a GABAA response. It is thus possible to block it selectively by applications of bicucullinc mcthiodide or picrotoxin (10/~M): GABAA antagonists are not very useful as tools in the dissection of cortical synaptic potentials, since blockade of the fast IPSP is quickly followed by burst-like cpileptiform activity (Connors et al., 1988; Tseng and Haberly, 1988). Similarly, the fast IPSP of rat ncocortex is reduced in amplitude by 1.7 mM penicillin before the onset of cpilcptiform activity: the time-to-peak of the fast IPSP is also prolonged by penicillin (Weiss and Hablitz, 1984). The pharmacology of the fast IPSP has been extensively investigated by Scholficld, who studied the effects of several GABA-relatcd drugs on the conductance change associated with the fast IPSP in guinea pig olfactory pyramidal cells in vitro (1980a,b; Riley and Scholficld, 1983). Bicuculline (1 # M) was the most potent antagonist tested, causing a marked reduction in the fast IPSP. Higher concentrations were required for picrotoxin (100#M), bemegride (30 #M-1 mM) or pentylenetetrazol (2 mM). Non-specific antagonistic effects are also observed with the nicotinic antagonist d-tubocurarine (30 #M) or the glycine antagonist strychnine (10-100 #M) at concentrations several orders of magnitude higher than those that can be expected to be selectively active against acetylcholine or glycine, respectively (Scholfield, 1980a). Barbiturates enhance and prolong thc fast IPSP and thc associated conductance increase in the olfactory cortex (Scholfield, 1978b, 1980b; Tseng and Haberly, 1988) as well as in the neocortex (Weiss and Hablitz, 1984). Pentobarbitone (20-200#M) has a selective effect on the fast IPSP, while in higher concentrations it also decreases neuronal input resistance and EPSP amplitude. Phenobarbitone is 10 times less potent than pentobarbitone, whereas methobexitone is 10 times more potent (Scholfield, 1980b). The effect of barbiturates is very pronounced, with potentiations of the fast IPSP conductance increase of up to 50-fold: note, however, that accurate measurements of this conductance change are complicated by the overlap with the EPSP and by the sheer size of the conductance increase itself (Scholfield, 1978b). Other general anaesthetics increase selectively the duration of the conductance change during the fast IPSP: Scholfield (1980b) has reported a very marked prolongation (10-fold or more) by chloralose and alphaxalone and smaller effects (up to 2-fold prolongation) by halothane, ketamine and urethane. The concentration ranges for the selective effects were 5-100#M for chloralose, 0.2-10 #M for alphaxalone, 0.1-2 mM for halothane, up to 0.5 mM for ketamine and 10-50 mM for urethane (Scholfield, 1980b). The duration and amplitude of the conductance change during the fast IPSP are also increased by diazepam at the rather high concentration of 30 #M (Riley and Scholfield, 1983). The slow IPSP lasts several hundreds of milliseconds and is associated with a conductance increase smaller than that underlying the fast IPSP in both olfactory cortex and neocortex slices (Tseng and Haberly, 1988; Connors et al., 1982, 1988; Avoli, 1986; Howe et al., 1987a). The latency of the slow IPSP is difficult to measure, since its onset is obscured

67

by the EPSP and the fast IPSP. Fairly low values (less than l0 ms) have been reported for olfactory cortex neuronvs during fast IPSP blockade (Tseng and Haberly, 1988). The slow IPSP is fairly labile, decreasing markedly at stimulation frequencies above 0.125--0.2 Hz: twenty stimuli at 1 Hz are sufficient to cause complete fatigue in the associated conductance increase (Connors et al., 1982; Howe et aL, 1987a). While anomalous rectification of the pyramidal cell membrane can prevent reversal of the slow IPSP during injection of hyperpolarizing current (Avoli, 1986), all studies agree in finding the slow IPSP reversal potential more negative than the resting membrane potential, with values around - 9 0 mV (Connors et al., 1982, 1988; Tscng and Haberly, 1988). The reversal potential for the slow IPSP is similar to that of the afterhyperpolarization which follows a train of action potentials (Howe et at, 1987a) and is markedly affected by changes in the external potassium concentration (Avoli, 1986; Howe et aL, 1987a; Tseng and Haberly, 1988). The slow IPSP appears to be mediated by GABAB receptors. In fact, its reversal potential is similar to that for the neuronal responses to GABA (in the presence of bicuculline methiodide) or for baclofen (Connors et al., 1988). The slow IPSP is not affected by 10-50 pM bicucuiline (Howe et aL, 1987a): slight increases have been observed (Connors et al., 1988) which may be due to a reduction in the shunting effect of the slow IPSP by the fast IPSP. Finally, application of 1 mM phaclofen to the neocortical slice quickly and reversibly suppresses the slow IPSP (Karlsson et aL, 1988; Karlsson and Olpe, 1989). In the olfactory cortex Tseng and Haberly (1988) have found that, while the amplitude of the fast IPSP responds more or less linearly to depolarization from the resting membrane potential, the amplitude of the slow IPSP at first increases and then decreases with depolarization, reaching a maximum around - 5 0 i n V . It has been reported (Howe et aL, 1987a) that in the neocortex the slow IPSP could no longer be detected if the membrane potential was depolarized to values positive to - 5 5 mV; membrane depolarization also markedly affects the duration of the slow IPSP and the duration of the underlying conductance change (assessed as the period during which the IPSP blocks action potentials generated by direct current injection). At the peak of the slow IPSP, the conductance increase (expressed as percent change with respect to the prestimulus value) does not change significantly with membrane potential (Howe et al., 1987a). Data on the voltage-dependence of the slow IPSP should however be evaluated with caution, in view of the non-linearity in the membrane properties of cortical neurones and the dendritic generation of this synaptic potential (see below). There is little doubt that the slow IPSP has a synaptic origin and it is not an intrinsic voltage-dependent response (e.g. activation of a Ca:+-depen dent K + conductance) since it is insensitive to blockers of Ca2+-activated K + currents, such as 8-bromo-cAMP and intracellular EGTA. Both the fast and the slow IPSP are readily blocked by low Ca2+-high Mg :+ media, suggesting that they are di- or polysynaptic responses, whereas the EPSP is monosynaptic (Scholfield, 1978a; Tseng and

68

L. SIVILOTTIand A. NISTR1

Haberly, 1988). As for the location of the synapses that generate the IPSPs, the selective blocking effects of dendritic or somatic application of Cd 2+ suggest that many of the synapses subserving the fast IPSP are near the cell body, while those involved in the slow IPSP are mostly on proximal dendrites (Tseng and Haberly, 1988). The identity of the neurones responsible for the generation of these IPSPs is not entirely clear. In fact, Satou et al. (1983b) have suggested that some deep interneurones may produce IPSPs in the pyramidal cell via a feedback circuit. Nevertheless, normal synaptic responses to lateral olfactory tract stimulation can still be elicited in slices from which layer III has been removed (Tseng and Haberly, 1988). Since this lesion entails the disruption of pyramidal cell axons with their recurrent collaterals and the loss of deep interneurones, it appears that the fast and the slow IPSPs are mediated by feed-forward mechanisms involving superficial interneurones activated by lateral olfactory tract stimulation. 7.2.3. GABAergic synaptic potentials o f interneurones The electrophysiology of some GABAergic neocortical neurones (corresponding to aspiny or sparselyspiny cells) has been described by McCormick et al. (1985): IPSPs in response to synaptic stimulation were observed in less than half of the cells sampled. In the olfactory cortex deep, presumptively inhibitory neurones in layer III may respond to olfactory bulb stimulation with a complex pattern consisting of depolarization (with action potential), brief hyperpolarization, depolarization (with a burst of action potentials) and hyperpolarization (Satou et al., 1982b, 1983b). While the last hyperpolarization resembles the slow IPSP recorded from principal neurones, the technical difficulty of recording intracellularly from interneurones for prolonged periods precluded the possibility of proving that this potential is synaptic in origin rather than being generated by a slow intrinsic neuronal conductance. 7.3. OLFACTORYBULB 7.3.1. GABAergic pathways Immunocytochemical visualization of GAD in the olfactory bulb reveals a radial gradient of staining, starting from intense reactivity in the glomeruli and in the external plexiform layer (particularly in the superficial half) and displaying a lower density of neuropil staining in deeper layers (Ribak et al., 1977; Mugnaini et al., 1984a,b). The stained processes in the external plexiform layer line up adjacent to the secondary dendrites of the mitral cells: electron microscopy shows that these structures correspond to the gemmules or spines of the granule cells (or, in the glomeruli, the periglomerular cells) which form reciprocal dendro-dendritic synapses with the mitral cells (and the tufted cells in the glomeruli; Ribak et al., 1977). Putative GABAergic neurones belong to all the morphological types of intrinsic olfactory bulb neurones, whereas principal cells (e.g. mitral and tufted neurones) do not appear to contain GABAergic

markers (Ribak et al., 1977). GABAergic neurones therefore include periglomerular, granule and shortaxon cells. Periglomerular cells contain GAD (Ribak et al., 1977; Mugnaini et al., 1984a,b), newly-synthesized GABA-T (Nagai et al., 1983) and GABA (particularly in periglomerular areas; Ottersen and StormMathisen, 1984; Gall et al. 1987). About 70% of periglomerular cells stain for both GAD (or GABA) and tyrosine hydroxylase and may therefore release both GABA and a catecholamine from their synaptic terminals (Kosaka et al., 1985, 1987; Gall et al., 1987; note the contrasting findings of Mugnaini et al., 1984b). GABAergic markers are also present in granule cells, reaching particularly high levels in their dendritic processes (including the spines or gemmules; Ribak et al., 1977; Halfisz et al., 1979; Nagai et al., 1983; Mugnaini et al., 1984a; Ottersen and StormMathisen, 1984; Gall et al., 1987). Finally, many short-axon cells in the external plexiform layer show immunoreactivity for GAD or GABA (76%; Gall et al., 1987; see also Mugnaini et al., 1984a,b). Approximately half of GABAergic short-axon cells also contain tyrosine hydroxylase (Gall et al., 1987). Kosaka et al. (1987) have also reported that a small proportion of GAD-immunoreactive cells of unspecified morphology in the granule cell layer also stain for the neuropeptide Met-enkephalin. GABA-containing fibres have been described in the olfactory nerve layer (Ottersen and Storm-Mathisen, 1984), but their origin is unclear. Some nerve terminals labelled by [3HI GABA may originate from the anterior olfactory nucleus (Hai~isz et al., 1979). It should be noted that deafferentation does not affect the bulbar levels of GABAergic markers and, additionally, that the contribution--if any--of centrifugal bulbopetal GABA fibres is probably small (see the review by Hal~tsz and Shepherd, 1983). 7.3.2. GABAergic synaptic potentials The membrane and synaptic properties of olfactory bulb neurones have been recently reviewed by Mori (1987). 7.3.2.1. Spontaneous events Spontaneous IPSPs have been described only in the in vitro olfactory bulb preparation of the turtle (Mori et al., 1981a; Jahr and Nicoll, 1982). These potentials

appear as small hyperpolarizing fluctuations in the membrane potential of mitral cells: these events revert to depolarizing when the cell is hyperpolarized by current injection. Their GABAergic origin is proved by their sensitivity to bicuculline (0.1 mM) and their polarity reversal after intracellular C1- loading. The spontaneous IPSPs are abolished by TTX and greatly increase in frequency following antidromic or orthodromic stimulation (Jahr and Nicoll, 1982). 7.3.2.2. Evoked potentials The occurrence of hyperpolarizing synaptic potentials in rabbit mitral cells in vivo was reported

GABA R£CEPa'ORMECHANISMS

independently by Phillips et al. (1963) and Yamamoto et al. (1963). IPSPs can be produced in mitral neurones by stimulation of their axons in the lateral olfactory tract (Phillips et al., 1963; Yamamoto et al., 1963; Mori and Takagi, 1978a) or by stimulation of the olfactory nerve (Yamamoto et al., 1963; Getchell and Shepherd, 1975) or the anterior commissure (Mori and Takagi, 1978b). The second IPSP that follows antidromic stimuli has a lower threshold than the spike (Yamamoto et al., 1963; Mori and Takagi, 1978a), lasts on average 300 ms in the rabbit in vivo and is associated with a decrease in input resistance (Mori and Takagi, 1978a). This synaptic potential displays a complex behaviour in response to changes in membrane potential brought about by current injection: in fact, the earlier part of the IPSP reaches its reversal potential at lower intensities of hyperpolarizing current than the later part; Mori and Takagi (1978a) have inferred that the synapses that generate the earlier part of the IPSP are, on the whole, electrotonically closer to the cell body and the recording microelectrode. Similar properties have been described for the IPSP evoked by anterior commissure stimulation, suggesting that both potentials may be generated by activation of the same set of synapses on the mitrai cell dendrites (Mori and Takagi, 1978b). The orthodromic IPSP elicited by olfactory nerve stimulation is a large hyperpolarizing potential with a complex time-course at the low value of recorded membrane potential (Getchell and Shepherd, 1975). The difficulty in obtaining stable intracellular recordings in the intact animal has precluded the full pharmacological characterization of the IPSP. Strong evidence supporting the GABAergic nature of the IPSP has come from investigations into the extracellular correlates of the IPSP in civo and from intracellular studies of in vitro non-mammalian preparations. Extracellularly, mitral cell IPSPs are reflected by an inhibitory pause in mitral cell firing produced by lateral olfactory tract or olfactory nerve stimuli in the rabbit or cat in vivo: surprisingly, i.v. bicuculline administration has no effect or prolongs this synaptic inhibition (Felix and McLennan, 1971; McLennan, 1971). On the other hand, another correlate of mitral inhibition, the paired-pulse depression of antidromic field potentials, is blocked by i.v. bicuculline or picrotoxin (McLennan, 1971; Nicoll, 1971). Intracellular studies in vitro have been carried out in the hemisected olfactory bulb preparation of the turtle. Mitral cells in this preparation respond with complex inhibitory potentials to lateral olfactory tract or olfactory nerve stimulation (Mori et al., 1981a,b; Jahr and Nicoll, 1982). Mori et al. (1981a,b) have reported the presence of three distinct hyperpolarizing components in these synaptic responses, which were termed I~,/2 and Is and were very similar for both types of stimuli. The first two components have a threshold lower than either the anti- or the orthodromic action potential, are graded and behave linearly in response to membrane potential changes, with reversal potentials approximately 15 mV negative to the resting value (Mori et al., 1981a). Note, however, that in cells with more hyperpolarized membrane potentials ( - 7 5 to - 8 5 mV) I 1 and I: are

69

depolarizing. Due to the fast time course of I~, a fall in input resistance can be reliably demonstrated only during I 2. These two hyperpolarizing components appear to be GABAergic IPSPs, since they are blocked by 10/~M bicuculline, reversed by intracellular C1- loading and virtually abolished by replacement of CI- in the extracellular medium with propionate or glutamate (Nowycky et aL, 1981). The origin of the third hyperpolarizing potential remains obscure: despite the anomalous voltage-dependence of I s (whose amplitude decreases with either depolarization or hyperpolarization from resting membrane potential and cannot be reversed), a mechanism other than the activation of GABAB synaptic receptors is likely, since this potential is not associated with a demonstrable conductance increase and is suppressed by any manoeuvre that reduces or indeed reverses I l and I 2 (Mori et al., 1981b; Nowycky et aL, 1981). In the same preparation Jahr and Nicoll (1982) have reported that a Cl--dependent IPSP (associated with a conductance increase) is generated after the stimulation of the lateral olfactory tract or the olfactory nerve, or after the direct activation of mitral cells by depolarizing pulses. While the IPSP produced by direct or antidromic stimulation is abolished by picrotoxin or bicuculline (0.1 raM), a residual synaptic hyperpolarization persists in the presence of GABA antagonists following olfactory nerve stimulation. This hyperpolarization (and its small conductance increase) might involve dopaminergic synapses (Jahr and Nicoll, 1982) or GABA B receptors. The similarity of IPSPs elicited by antidromic or orthodromic stimulation has been taken by Mori et aL (1981 a) as an indication that GABAergic synapses on the secondary (rather than the primary) dendrites of mitral cells are involved. In fact, should primary dendrites participate in the genesis of the IPSP recorded from the soma, differences in anti- and orthodromic responses would be expected, since the orthodromic input from the olfactory nerve terminates only on primary dendrites. The sequence of events in the production of IPSPs evoked by direct, anti- or orthodromic stimulation includes mitral dendritic depolarization, activation of the excitatory dendro-dendritic mitral-granule cell synapse, granule cell dendritic depolarization and activation of the dendro-dendritic granule-mitral GABAergic synapse. This was demonstrated with field potential analysis by Rall and Shepherd (1968) and is supported by Jahr and Nicoll (1982), who have shown that a great part of the IPSP is a consequence of action potential invasion of mitral cells. When somatic invasion by an antidromic spike is prevented by hyperpolarizing the cell so that only an initial segment spike is present, the IPSP is much reduced and is probably generated by the recurrent collaterals of mitral cell axons (Jahr and Nicoll, 1982). Mori et al. (1983) have reported that some tufted cells (identified as such by horseradish peroxidase injection) respond with small IPSPs to lateral olfactory tract stimulation in the rabbit in vivo. In the rat in vivo the response of tufted cells to olfactory nerve shocks consists of an EPSP (with spikes) followed by an IPSP (Schneider and Scott, 1983). No hyperpolarizing potentials have been observed in granule cells in

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response to lateral olfactory tract or anterior commissure stimulation (Mori and Takagi, 1978b). Nevertheless, the instability of most examples of intracellular recordings from tufted and granule cells and, hence, the small numbers sampled make it difficult to draw general conclusions.

7.4. SEPTALNUCLEI 7.4.1. G A B A e r g i e pathways Staining for the presence of G A D reveals intense immunoreactivity in the neuropil of the lateral septal nucleus, whereas the density of GAD-positive nerve terminals is only weak to moderate in other septal areas, including the medial septum, the nucleus of the diagonal band of Broca and the bed nucleus of the stria terminalis (Panula et al., 1984). As for GABAergic somata, groups of putative GABAergic neurones are present throughout the septal areas and belong to two distinct classes of cells: a group of small neurones in the lateral septai nucleus (especially in the pars dorsalis) and a population of larger cells in the nucleus of the diagonal band of Broca and in the medial septum (Panula et aL, 1984). Additionally, GABA-T-reactive neurones can be observed in the bed nucleus of the stria terminalis and in the substantia innominata (Nagai et al., 1983). Most or all of the GABAergic terminals in the septal area are likely to originate from intrinsic nuclei, since lesions of afferent pathways do not affect septal concentrations of GABAergic markers (Fonnum et al., 1977; Storm-Mathisen and Opsahl, 1978). Low to moderate levels of G A B A A and GABAB binding sites have been demonstrated in the medial septum (Bowery et al., 1987).

7.4.2. G A B A e r g i e synaptie potentials 7.4.2.1. Spontaneous events Hyperpolarizing spontaneous IPSPs have been described in rat lateral septal neurones in vitro: these potentials are blocked by bicuculline methiodide or picrotoxin and have a reversal potential of - 7 1 mV (Stevens et al., 1987). Picrotoxin-sensitive spontaneous depolarizing potentials can be observed in rat medial septal nucleus neurones in vitro for up to 10-12 hr after the preparation of the slice (Segal, 1986).

7.4.2.2. Evoked potent&& The occurrence of an IPSP in lateral septal neurones was first described by DeFrance et al. (1972) in the rat/n v&o: this hyperpolarizing potential is part of an EPSP-IPSP sequence evoked by stimulation of the ipsilateral fimbria, is graded and can be elicited by stimuli which are subthreshold for spike generation. Extracellularly, this response is reflected by activation of single unit firing following by prolonged inhibition (McLennan and Miller, 1974a). The GABAergic

nature of the mechanisms which underlie this synaptic inhibition is supported by the substantial shortening of the inhibitory pause brought about by the iontophoresis of bicuculline methochloride, but not of strychnine (McLennan and Miller, 1974b; DeFrance et al., 1975). An EPSP-IPSP sequence can also be evoked in the lateral septum by stimulation of ventral septal afferents: this pathway seems to activate the same inhibitory interneurones which mediate fimbrial inhibition, since the inhibition from the two types of stimulation does not summate (DeFrance et al., 1976). In rat lateral septal neurones in vitro, IPSPs can be evoked by stimulation of the fimbria, the fornix, the medial septum or by local stimulation (Stevens et al., 1987). At higher stimulation intensities, the IPSP component of the EPSP-IPSP sequence becomes clearly biphasic, and consists of a fast and a slow component. The fast IPSP is blocked by 10 ~tM bicuculline methiodide or picrotoxin and has a reversal potential of - 7 4 mV, whereas the slow IPSP is unchanged or enhanced by bath applications of G A B A A antagonists and has a reversal potential of - 9 3 mV (Stevens et al., 1987). The mediation of the slow IPSP mainly by a GABAB receptor mechanism is suggested by its sensitivity to 0.5 mM phaclofen (Hasuo and Gallagher, 1988). In the medial septum, fimbrial stimulation elicits antidromic activation and an inhibitory response: the latter consists of either a long lasting suppression in spontaneous firing or a transformation of the spontaneous random activity into a periodic bursting discharge (McLennan and Miller, 1974a). Both types of inhibition are disrupted by bicuculline methochloride (McLennan and Miller, 1974b). Dutar et al. (1985) have shown with intracellular recording from the medial septum and the nucleus of the diagonal band of Broca of the rat in vivo that fimbrial shocks produce an antidromic spike which is followed in most neurones by an EPSP and in all cells by an IPSP. This IPSP is sensitive to C1 injection and is associated with a conductance increase. Differences in the time course of the response to intracellular CI- loading by the early and late parts of the IPSP could indicate the presence of more than one inhibitory component or the spread of the GABAergic synapses over a wide area of the neurone (Dutar et al., 1985). Segal (1986) has described a depolarizing IPSP evoked in rat medial septal neurones in vitro by lateral septum stimulation: this potential is CI -dependent, has a reversal potential of - 6 0 mV and is suppressed by microdrop applications of 5mM picrotoxin. The presence of this GABA A antagonist fails to unveil any residual hyperpolarizing synaptic response. The neuronal circuits which subserve inhibition in the septum are likely to be complex: extracellular data indicate that feed-back mechanisms may operate in the lateral septum (including its dorsal part; McLennan and Miller, 1974a; DeFrance et al., 1975). In the circuit diagram put forward by McLennan and Miller (1974a), feed-forward inhibition from the fimbria is present only in the medial septum. DeFrance et al. (1975) have however suggested that this type of mechanism may operate also in the dorsal part of the lateral septum.

GABA RECEPTORMECHANISMS 7.5. THALAMUS 7.5.1. GABAergic pathways GABAergic markers are present in the neuropil of all thalamic areas, but their density can vary. A sparse, low density of staining for GAD, GABA or newly-synthesized GABA-T has been reported for the nucleus reticularis (Houser et al., 1980; Hendrickson et al., 1983; Nagai et al., 1983; Ottersen and StormMathisen, 1984). These presumptive GABAergic terminals sometimes form synapses on GABAergic neurones (Houser et al., 1980; Ottersen and StormMathisen, 1984). Practically all cells in this nucleus contain GAD, GABA or GABA-T (Houser et al., 1980; Oertel et al., 1983; Hendrickson et al., 1983; Nagai et al., 1983; Ottersen and Storm-Mathisen, 1984) and are one of the sources of GABAergic inhibition to the thalamus proper (Steriade and Deschrnes, 1984; Jones, 1985): indeed, numerous GABA-containing fibres in the thalamus can be observed near the nucleus reticularis (Ottersen and Storm-Mathisen, 1984). Most (perhaps all) the neurones in the cat nucleus reticularis display somatostatin immunoreactivity, suggesting that this peptide may be used as a co-transmitter with GABA (Oertel et al., 1983). The co-localization of GABA with neuropeptides may however be species dependent, since no somatostatin immunoreactivity is found in the rat nucleus reticularis (Finley et al., 1981). GAD-positive terminals have been described in the neuropil of several thalamic nuclei (Houser et al., 1980). These terminals originate from local circuit neurones, from the nucleus reticularis and from extrathalamic afferents. In fact, GABAergic structures such as the substantia nigra and the pallidus-entopeduncular nucleus are known to project to the ventromedial and ventroanterolateral thalamus, respectively (DiChiara et al., 1979; Penney and Young, 1981). In the thalamus proper, GABAergic perikarya are far fewer than in the nucleus reticularis: approximately 20-30% of the total cell population were GAD-positive neurones in the ventral posterolateral and ventral posterior nucleus of the cat (Spreafico et al., 1983; Penny et al., 1983). The same authors reported also that these cells are smaller in size than the relay thalamocortical neurones (see also MadarS.sz et al., 1985) and that they do not project to the cortex. Somata immunoreactive for GABA are unevenly distributed in the thalamus: some are found in the lateral posterior and ventromedial nuclei, but very few are present in the ventrobasal complex and none in the anterior and medial thalamic nuclear group (Ottersen and Storm-Mathisen, 1984). Using a similar approach, Madarfisz et al. (1985) have estimated that GABA-containing cells represent 33 and 25% of the total neuronal population in the cat ventrobasal complex and anteroventral nucleus, respectively. Neurones positive for newly-synthesized GABA-T appear to be localized in distinct thalamic regions: high densities are present in the midline, intralaminar and reticular nuclei with unstained areas in between (Nagai et al., 1983). In the lateral geniculate, GAD reactivity is localized in a variety of structures, which include glomeru-

71

lar profiles, puncta and intrinsic cells (Hendrickson et al., 1983). The highest neuropil staining is found in laminar areas (Hendrickson et al., 1983). A detailed review of the vast literature on the ultrastructure, origin and synaptology of GABAergic terminals in the lateral geniculate is outside the scope of the present work (for a comprehensive discussion see Sherman and Koch, 1986). Briefly, GABAergic markers are found in glomerular profiles (F2): these are the dendrites of intrinsic neurones which receive synapses from optic tract afferents and from other F 2 terminals, and in turn establish synapses on relay geniculate cells and other F 2 dendrites (Sterling and Davis, 1980; Hendrickson et al., 1983; Montero, 1986). Additional extraglomerular GABAergic terminals derive from the axons of perigeniculate afferents and of intrinsic GABAergic neurones: these synapse on relay neurones and on GAD-positive structures (Hendrickson et al., 1983; Ohara et al., 1983). GABAergic somata are evenly distributed in laminar and interlaminar geniculate areas: they represent approximately 25% of geniculate cells, are local circuit neurones and have a smaller perikaryon than relay neurones (Sterling and Davis, 1980; Hendrickson et al., 1983; Ottersen and Storm-Mathisen, 1984; Fitzpatrick et al., 1984; Madarfisz et al., 1985). Nagai et al. (1983) have reported that GABA-T positive cells occur in both the ventral and the dorsal division of the lateral geniculate, whereas no GAD-positive cells have been detected by Hendrickson et al. (1983) in the ventral lateral geniculate. In the perigeniculate nucleus, which may be considered a division of the nucleus reticularis, all cells are GABAergic (Fitzpatrick et al., 1984; Montero and Singer, 1984). The ultrastructure of GAD-positive terminals in this area has been described (Montero and Singer, 1984). Fairly high levels of muscimol binding sites have been observed in the lateral and medial thalamus (Unnerstall et al., 1981); the distribution of A and B binding sites has been investigated in more detail by Bowery et al. (1987), who have reported particularly high concentrations of both receptor subclasses in ventroposterior, ventromediai, ventrolateral and laterodorsal areas and in the lateral geniculate. It is worth noting also that in the lateral posterior nucleus GABA~ binding sites are more abundant than GABAA and that the ventral lateral geniculate has low levels of both.

7.5.2. GABAergic synaptic potentials Following the early intracellular demonstration of Cl--dependent IPSPs evoked in ventrobasal or lateral geniculate neurones by orthodromic or local stimulation (Purpura and Cohen, 1963; Andersen et al., 1964d; Burke and Sefton, 1966), a wealth of evidence has accumulated on the role of GABAergic inhibition in determining the functional properties of thalamic cells (for reviews see Steriade and Desch~nes, 1984; Jones, 1985; Sherman and Koch, 1986; Martin, 1988). In the present survey we shall focus on the pharmacological properties of thalamic GABAergic IPSPs with particular reference to intracellular studies in vitro.

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L. SIV1LOTTIand A. NISTm

Pharmacological evidence for a role of GABA in thalamic inhibition was first obtained in the cat in vivo by Curtis and TebEcis (1972), who found that the depression of lateral geniculate neuronal firing induced by stimulation of the cerebral cortex or of the optic nerve is blocked by bicuculline but not by strychnine. Similarly, the inhibition of cell firing produced in the ventrobasal thalamus by stimulation of the primary somatosensory cortex is sensitive to bicuculline. In coronal slices of the rat thalamus, local stimulation evokes, in posterior relay neurones, EPSPs which may be followed by IPSPs (Thomson, 1988a); in more rostrally cut slices, which contain the greater part of the nucleus reticularis, stimulation of this nucleus elicits mostly pure IPSPs a n d - - i n less than 30% of neurones--an EPSP-IPSP sequence (Thomson, 1988a). The IPSP is, in most neurones, a depolarizing potential: its reversal potential of - 6 6 mV is fairly close to the resting membrane potential and is shifted to more depolarized levels by intracellular CI injection (Thomson, 1988a,b). It must be noted, however, that the late phase of the IPSP has a more depolarized reversal potential ( - 52 mV) and that, at appropriate membrane potentials, biphasic hyperpolarizing-depolarizing signals can be observed. The IPSP is associated with a conductance increase which can be very marked. The IPSP is antagonized by somatic iontophoresis of bicuculline methobromide or picrotoxin, although the late component of this potential is more sensitive than the early one (Thomson, 1988a). Further confirmation of the GABAergic nature of this signal comes from the observation by Sykes and Thomson (1989) that pentobarbitone can increase the duration and amplitude of the IPSP and can enhance the underlying conductance increase together with a depolarizing shift in the reversal potential. In dorsal lateral geniculate relay neurones in vitro, stimulation of the optic tract produces an EPSP-fast IPSP-slow IPSP sequence: essentially similar responses have been described in cat or rat slices, regardless of the preservation of the reticular or perigeniculate nucleus in the slice (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1988, 1989a). In cat geniculate slices, neurones which display this synaptic response have been identified as belonging to a variety of morphological types, including subclasses of X and Y relay cells and are distributed among laminae A~, A 2 and C (Soltesz et al., 1989a). Among the synaptic potentials of the geniculate the EPSP has the highest threshold, while the fast and slow IPSPs are elicited by similar stimulus intensities (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1989a). The properties of both inhibitory potentials may suggest that they are seemingly generated via a disynaptic circuit (Crunelli et al., 1988). Furthermore, the fast IPSP fatigues at frequencies of stimulation higher than 0.3-0.5 Hz and the slow IPSP at frequencies above 0.05 Hz, with a maximal depression for the latter of 20--40% at 0.5 Hz (Hirsch and Burnod, 1987; Crunelli et aL, 1988; Soltesz et al., 1989a). Slow IPSPs recover from the depression induced by a conditioning optic tract

shock with a time course much slower than EPSPs (Hirsch and Burnod, 1987). The fast IPSP has all the ionic and pharmacological properties expected of a GABA a receptor-mediated response: it is a short response (34 ms at 35°C), blocked by picrotoxin or bicuculline methiodide (or methobromide), but not by high concentrations of phaclofen (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1988, 1989a). Furthermore, the fast IPSP is associated with a marked conductance increase and has a reversal potential ( - 6 8 mV in control conditions) sensitive to intracellular CI- injection or to changes in extracellular CI concentrations. Finally, its behaviour in response to changes in membrane potential induced by d.c. current injection is fairly linear between - 4 5 and - 7 5 m V (Crunelli et al., 1988; Soltesz et al., 1988, 1989a). The slow IPSP is a graded potential, which lasts 240-300 ms and can often be evoked in isolation (in 50% of the cells in the sample described by Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1989a). This response appears to be generated by activation of synaptic receptors and not by intrinsic neuronal conductances, since it cannot be elicited by depolarization per se and is unaffected by intracellular Ca 2÷ chelators such as EGTA (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1989a). Far from being blocked by G A B A A antagonists, slow IPSPs are actually enhanced by 50/~M picrotoxin or by lO-50#M bicuculline (Hirsch and Burnod, 1987; Soltesz et al., 1989a), presumably because any preceding fast IPSP component is removed by these antagonists or because of disinhibition of GABAergic interneurones. The slow IPSP is also enhanced by the GABA uptake inhibitor nipecotic acid (1-10 p M) in the presence of bicuculline methiodide (Crunelli et al., 1988) and is antagonized by the G A B A 8 competitive blocker phaclofen (0.5-1 mM) in the presence of bicuculline methiodide (Soitesz et al., 1988). A conductance increase has been shown to be associated with the slow IPSP, but it is more modest than the one observed during the fast IPSP (Crunelli et al., 1988). The amplitude of the slow IPSP does not behave linearly in response to membrane potential changes, but is maximal at resting potential and decreases with both membrane hyperpolarization and depolarization (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et aL, 1989a). Notwithstanding the voltage-dependence of the GABAB-activated conductance, this phenomenon could also be explained by the strong membrane rectification observed in these neurones: in fact, the slow IPSP still exerts an inhibitory action at depolarized membrane potentials when its amplitude is extremely small (Hirsch and Burnod, 1987). As a result of membrane inward rectification, a clear reversal of the slow IPSP was obtained only in 50% of the neurones in the sample described by Crunelli et al. (1988): in this study, the extrapolated value of the reversal potential was - 76 mV. A similar value ( - 8 5 mV) has been reported by Crunelli and co-workers (Soltesz et al., 1989a) for cat lateral geniculate neurones with an external K ÷ concentration of 5 mM. Hirsch and Burnod (1987), who worked on cat geniculate slices have found a - 104 mV reversal potential with an external K ÷ level

GABA RECEPTORMECHANISMS of 2 mM. The slow IPSP reversal potential responds to changes in the external K + concentration with shifts predicted by the Nernst equation for a K÷-de pendent response and is unaffected by intracellular C1- loading (Hirsch and Burnod, 1987; Crunelli et al., 1988; Soltesz et al., 1989a). The slow IPSP is blocked by 1 mM Ba 2÷ (see Section 4.2; Crunelli et al., 1988). In the cat, stimulation of putative corticogeniculate fibres evokes a synaptic response similar to that which follows optic tract stimuli: Soltesz et al. (1989a) have however observed that it is difficult to obtain a pure cortico-geniculate stimulation without antidromic activation of relay geniculate cells or activation of the perigeniculate nucleus. The circuits underlying IPSP generation in the lateral geniculate in the intact animal are likely to be several, involving both feed-forward and feed-back pathways (Sherman and Koch, 1986). Feed-forward inhibition is likely to originate from excitation by optic tract afferents of local geniculate interneurones. The GABAergic structures involved are both the axonal terminals and the dendrites of these interneurones: these dendrites are in fact presynaptic to geniculate relay cells (see Section 7.5.1). Furthermore, several feed-back loops can be activated by excitation of geniculate relay neurones and their recurrent collaterals: these may form excitatory connections with GABAergic neurones within the lateral geniculate itself or in the perigeniculate or may turn on longer feed-back loops involving the cerebral cortex. In any case, the in vitro evidence indicates that intrageniculate interneurones themselves can mediate both GABAA and GABAs potentials: a possible wiring diagram for intrageniculate inhibition has been put forward by Soltesz et al. (1989a). Hirsch and Burnod (1987) have suggested that the fast IPSP may be generated by feed-forward mechanisms because it has a latency locked to that of the EPSP and a lower threshold than the EPSP itself. The hypothesis that inhibition is mainly feed-forward for X cells and feed-back for Y cells (Sherman and Koch, 1986) has not been supported by the recent work by Soltesz et al. (1989a). Crunelli and co-workers (Soltesz et al., 1989b) have also investigated GABAergic inhibition in the ventral lateral geniculate nucleus of the rat in vitro. This slice preparation does not preserve the nucleus reticularis. Optic tract stimulation evokes in putative relay neurones an EPSP-fast IPSP sequence: the fast IPSP has a lower threshold than the EPSP and fatigues at stimulation frequencies higher than 0.1 Hz. Fast IPSPs are reversibly blocked by 50/~M bicuculline methiodide, are associated with a very marked decrease in input resistance and have a reversal potential of - 6 3 to 67 mV. Sometimes optic tract stimulation results in the appearance of multiple bicuculline-sensitive small hyperpolarizing potentials which appear alone (with a long, variable latency) or follow the fast IPSP. The occurrence of spontaneous fast IPSPs which are sensitive to bicuculline methiodide and invert at - 7 0 mV has also been reported for these neurones. No slow, GABAs-mediated IPSP can be observed in this area, even in the presence of bicuculline or picrotoxin, despite the presence of GABA B receptors which can be activated by bath-applied baclofen. -

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Soltesz et al. (1989b) have suggested that while optic tract fibres may generate fast IPSPs by feed-forward circuits, GABAB receptors may only be activated by different inputs, perhaps of pretectal origin. The mechanism by which the multiple fast IPSPs are generated remains obscure, even if spillover of GABA from distant release sites has been hypothesized (Soltesz et al., 1989b).

7.6. STRIATUM,GLOBUSPALLIDUSAND ENTOPEOUNCULARNUCLEUS 7.6.1. G A B A e r g i c pathways In the striatum GAD immunocytochemistry reveals intense staining in the neuropil, except for the fibre bundles: the GABAergic terminals synapse onto dendrites, dendritic spines and somata of striatal neurones (Ribak et al., 1979; Oertel and Mugnaini, 1984). A large number of neostriatal neurones are GABAergic: GAD, GABA-T and GABA reactivity is present in medium-size (10-20pM) neurones with fusiform or round cell bodies, which are evenly distributed throughout the caudate-putamen and may represent more than 80% of the neuronal population of this area (Ribak et al., 1979; Nagai et al., 1983; Ottersen and Storm-Mathisen, 1984; Oertel and Mugnaini, 1984). A small number of medium-tolarge size neurones are also GAD-immunoreactive. Most of the striatal GABAergic neurones are projection cells which send their axons to the pallidus, the entopeduncular nucleus and the nigra, but local circuit neurones are also present. The latter represent 15% of the total cell population and belong to the medium-size aspiny cell type (Bolam et al., 1983), whereas projection cells are probably spiny neurones (Ribak et al., 1979). The vast majority, or perhaps the totality of GADcontaining neurones are immunoreactive for opioid peptides such as Met- or Leu-enkephalin, or substance P (Oertel and Mugnaini, 1984; Penny et al., 1986). The concentration of GABA binding sites in the striatum is low to moderate for both A and B types (Bowery et al., 1987). The globus pallidus-entopeduncular nucleus displays a very high density of GAD reactivity: GADpositive puncta outline GAD-negative somatic and dendritic profiles (Ribak et al., 1979; Ottersen and Storm-Mathisen, 1984). Electron microscopy shows GABAergic synapses on the somata and dendrites of pallidal neurones; most contacts are between GADpositive terminals and GAD-negative cells, since GABAergic somata appear to receive fewer GABAergic terminals (Ribak et al., 1979). A small number of medium-sized neurones which stain for GAD, GABA or GABA-T are also present in both areas (Ribak et al., 1979; Nagai et al., 1983; Ottersen and Storm-Mathisen, 1984). The majority of GABAergic terminals in the pallidus belong to extrinsic afferents from the striatum, rather than to local circuit neurones (Fonnum et al., 1978). The pallidus contains few GABA binding sites: interestingly, GABAB sites outnumber GABAA sites (Bowery et al., 1987).

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L. SIVILOTTIand A. NISTRI 7.6.2. G A B A e r g i c synaptic potentials

The most commonly observed synaptic response in striatal neurones in vivo is an EPSP-IPSP sequence (Buchwald et al., 1973): this can be elicited by stimulation of a variety of brain sites, which include substantia nigra, several thalamic areas (lateral posterior, ventrolateral, ventroanterior, centromedian and parafascicularis), cerebral cortex, dorsal raphe and striatum itself (Hull et al., 1970; Buchwald et al., 1973; VanderMaelen et al., 1979; for a review of the electrophysiology of responses evoked by the various afferent pathways, see Kitai, 1981). Stimulation of any of these sites evokes in a minority of cells apparently pure EPSPs, while isolated IPSPs are very rare (Buchwaid et al., 1973). The IPSP can last as long as 200-300 ms, particularly if trains of stimuli at frequencies higher than 10 Hz are used (Hull et al., 1970; Buchwald et al., 1973; VanderMaelen and Kitai, 1980); the amplitude of this hyperpolarizing IPSP decreases with hyperpolarization (Buchwald et al., 1973) but no data about its reversal potential in vivo are available. There is good evidence to support the mediation of at least part of the IPSP by GABA A receptors. Bernardi et al. (1976) observed that the IPSP component of EPSP-IPSP responses evoked by cortical or pallidal stimuli is abolished by i.v. administration of bicuculline or picrotoxin. Iontophoretic applications of bicuculline base or picrotoxin were ineffective, although this might partly reflect their insufficient delivery. Indeed, iontophoresis of bicucuiline methochioride was found by Herding (1984) to antagonize the hyperpolarizing IPSP evoked by cortical stimulation. Results similar to those of Bernardi et al. (1976) were described by Park et al. (1980) for nigrally-evoked responses. Herrling (1984) showed that bicuculline methochloride does not entirely suppress synaptic hyperpolarization, but spares a late component of it: this potential could be mediated by GABA B receptors or by neurotransmitters other than GABA, or by the activation of intrinsic neuronal conductances in the postsynaptic neurone. Alternatively, Wilson et al. (1983) have suggested that disfacilitation may underlie the long-lasting hyperpolarization seen in the caudate: nigral stimulation would thus hyperpolarize striatal cells by blocking the activity of tonic excitatory inputs from cortical and thalamic afferents. In support of this view, cortical ablation and thalamic transection suppress the long-lasting hyperpolarization (Wilson et al., 1983). The disfacilitation hypothesis rests also on the lack of a conductance increase associated with this hyperpolarization and on the anomalous behaviour of this potential in response to membrane potential changes (e.g. decrease with depolarization, increase with hyperpolarization). Note also that strong membrane rectification was observed (Wilson et al., 1983). When slice preparations are used, hyperpolarizing IPSPs are observed less commonly than in vivo. Indeed Misgeld and co-workers (1979, 1982) have failed to detect any hyperpolarizing response following the striatal EPSP evoked by local stimulation. Lighthall and Kitai (1983) have found IPSPs (following the locally-elicited EPSP) only in 17% of the neurones in their sample. In an earlier study, the same

group (Lighthatl et al., 1981) reported that IPSPs could be produced only when trains of local stimuli were applied, rather than single shocks. These IPSPs are graded and decrease in amplitude when the cell is hyperpolarized, but differ from their in vivo counterparts in that their duration is much shorter (30--40 ms; Lighthall et al., 1981; Lighthall and Kitai, 1983). Thus it has been suggested that extrastriatal circuits disrupted by slicing may mediate the longlasting inhibition of in vivo caudate neurones (Kitai and Kita, 1984). In neurones injected with the Na+-conductance blocker QX-314, Kita et al. (1985) have described an EPSP-IPSP sequence: the IPSP lasts for up to 130 ms in neurones depolarized by current injection and its reversal potential ( - 60 mV) is markedly depolarized by intracellular CI injection. This IPSP displays other GABAA-like properties as it is blocked by bicuculline or picrotoxin (both 50-200/~M) and enhanced by pentobarbitone (100/~M). Misgeld et al. (1982) have described a similar depolarizing IPSP with a reversal potential of - 5 7 to - 6 2 mV (cf. the reversal potential for GABA responses, - 5 1 mV): this potential can be observed only in slices treated with 100 #M pentobarbitone (with or without 50 t/M 4-aminopyridine). The depolarizing IPSP appears to speed up the decay of the preceding EPSP as shown by the prolonging effect of 50 ~M picrotoxin on the excitatory synaptic response. 7.7. SUBSTANTIANIGRA

7.7.1. G A B A e r g i c pathways The nigral neuropil shows very intense GADimmunoreactivity, particularly in the pars compacta, where GAD-positive puncta completely surround unstained neurones. Fewer GABAergic terminals (with a patchy distribution) are found in the pars reticulata (Ribak et al., 1976). Ottersen and StormMathisen (1984) found high levels of GABA-Iike immunoreactivity in the substantia nigra, with the highest density of terminals in the medial two-thirds of the pars reticulata. GABAergic synapses constitute the vast majority of synapses in this area: GABAergic terminals virtually cover the dendrites and (in the pars compacta) the somata of nigral neurones (Ribak et al., 1976). GABAergic neurones are also present in the substantia nigra, especially in the lateral aspect of pars reticulata (Nagai et al., 1983; Ottersen and StormMathisen, 1984). The cells of origin of the GABAergic terminals in the nigra are located in the striatum, particularly in the posterior part, and in the pallidus (McGeer et al., 1971; Hattori et al., 1973; Fonnum et al., 1974, 1978; Ribak et al., 1976; Smith and Bolam, 1989). Autoradiographic studies have detected high levels of GABA A and GABA a binding sites in the pars reticulata and moderate to low levels in the pars compacta (Bowery et al., 1987). 7.7.2. G,4BAergic synaptic potentials Data on inhibitory synaptic mechanisms activated in the nigra by striatal stimulation have been reviewed by Kitai (1981). The currently available


evidence has been obtained in in vivo experiments: the use of general or local anaesthesia, the different extent of cortical ablation and the choice of an enc6phale isol6 preparation by some authors may explain some of the reported discrepancies. Additionally, caudate stimulation may produce antidromic activation of nigral pars compacta neurones (Kitai, 1981; Collingridge and Davies, 1981). Thus, stimuli applied to the caudate can produce in the nigral neurones pure monosynaptic IPSPs (Yoshida and Precht, 1971) or combinations of EPSPs with shortand long-latency IPSPs (Frigyesi and Szabo, 1975). The extracellular correlates of the IPSPs evoked by striatal stimulation in the nigra are short- and longlatency inhibitions of single-unit firing (Precht and Yoshida, 1971; Collingridge and Davies, 1981) and a predominantly positive field potential (Precht and Yoshida, 1971). The caudate-evoked suppression of spontaneous discharges is mediated, at least in part, by GABA g mechanisms, since it is greatly reduced by i.v. picrotoxin in parallel with the depression of the field potential by this compound (Precht and Yoshida, 1971). Local iontophoresis of bicuculline methochloride (but not strychnine) is also effective in blocking a component of the synaptic inhibition of nigral firing (Collingridge and Davies, 1981): the possible contribution of GABA a mechanisms (Lacey et al., 1988) remains to be assessed. Further evidence on the GABAergic nature of IPSPs comes from the intracellular work by Grace and Bunney (1985) who showed that the IPSPs elicited by striatal stimulation in identified dopaminergic cells are sensitive to Cl- injection and to picrotoxin. Similar Cl--dependent IPSPs were also observed in non-dopaminergic neurones in pars reticulata, where they had a longer time course. 7.8. NUCLEUSACCUMBENS 7.8.1. GABAergic pathways High levels of G A D are present in this area (Walaas and Fonnum, 1979). Staining for newly-synthesized GABA-T reveals high neuropil levels and many positive cells: the latter are mostly small in size (Nagai et al., 1983). Fairly low concentrations of GABAA and GABAB binding sites have been detected in this area by Bowery et al. (1987). 7.8.2. GABAergic synaptic potentials In the accumbens of the rabbit in vivo a bicucullinesensitive component of the field potential is evoked by stimulation of the fimbria, which carries the hippocampal input to this area (DeFrance et al., 1985). More recently, Uchimura et al. (1989), working with an in vitro preparation of the guinea pig nucleus accumbens, reported the occurrence of GABAA-mediated IPSPs in response to local stimulation. The cells displaying this response are a subpopulation of neurones, which may belong to the large aspiny type described in this area. This IPSP was observed as part of an EPSP-IPSP sequence: it has a higher threshold than the EPSP and is reversibly and selectively

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blocked by 10/tM bicuculline or picrotoxin. At resting membrane potential, the IPSP is depolarizing, with a reversal potential ( - 3 4 mV) similar to that of neuronal responses to bath-applied GABA ( - 36 mV). 7.9. CEREBELLUM 7.9.1. GABAergic pathways The cerebellar cortex is an area particularly rich in GABAergic structures (for a review, see Nieuwenhuys, 1985). Staining for GAD reveals intense reactivity in puncta over the somata and the proximal dendrites of Purkinje cells and in the axons (and their terminals) of basket and stellate cells (Saito et al., 1974; McLaughlin et al., 1974; Ribak et al., 1978; Oertel et al., 1981). A similar pattern is seen for the distribution of GABA immunoreactivity, which is present in terminals on the Purkinje cell dendrites, in baskets around the Purkinje cell perikarya and in some structures in the cerebellar glomeruli which probably correspond to Golgi cell terminals. GABA-containing boutons appear to be more numerous in the molecular than in the granule cell layer (Ottersen and Storm-Mathisen, 1984). Roberts and co-workers (Kuriyama et al., 1966) have suggested that GABA is used as transmitter by Purkinje, basket, stellate and Golgi cells. Purkinje cells were the first neurones to be recognized as GABAergic in vertebrates (see the review by Krnjevir, 1982). Indeed, it has been reported that these neurones stain for GAD, albeit less intensely than other cerebellar somata (McLaughlin et al., 1974; Ribak et al., 1978; Oertel et aL, 1981). Colchicine treatment is necessary to obtain reproducible staining (Ribak et al., 1978). Chan-Palay et al. (1981) have however found that up to 40-50% of Purkinje cells (depending on the cerebellar region considered) are not GAD-immunoreactive; some of these cells contain the peptide motilin, while no known neurotransmitter has been detected in others. Approximately 10% of all Purkinje cells contain both motilin and GAD (Chan-Palay et al., 1981). Immunocytochemistry for GABA-like reactivity shows a wide range of staining intensities for Purkinje cells (Ottersen and Storm-Mathisen, 1984). These neurons are strongly reactive for newly-synthesized GABA-T, although occasional unstained or lightly-stained cells have been described (Nagai et al., 1983). The presence of GAD, GABA and newly-synthesized GABA-T has been reported also in basket, stellate and Golgi cells (McLaughlin et al., 1974; Ribak et al., 1978; Oertei et al., 1981; Nagai et al., 1983; Ottersen and Storm-Mathisen, 1984). On the basis of the distribution of high-affinity uptake processes for glycine and GABA, it has been suggested that Goigi cells comprise two distinct populations, which use GABA or glycine as transmitters (Wilkin et al., 1981). Other neurochemical evidence suggests that at least some stellate cells may release taurine (McBride and Frederickson, 1980). Deep cerebellar nuclei show numerous puncta positive for GAD or GABA which practically outline the GABA-negative somata and proximal dendrites of

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neurones in these nuclei (Saito et al., 1974; McLaughlin et al., 1974; Oertel et al., 1981; Ottersen and Storm-Mathisen, 1984). These terminals can be identified morphologically as the endings of Purkinje cell axons (McLaughlin et al., 1974): furthermore, lesions of Purkinje cell bodies result in a marked decrease in the level of GAD activity in this area (Fonnum et al., 1970). Indeed, bundles of GABA-positive fibres can be detected in the white matter between the cerebellar cortex and the deep nuclei (Ottersen and StormMathisen, 1984). The autoradiographical study by Chan-Palay (1978) has found laminar variations in the density of [3H] muscimol binding sites: levels are highest in the molecular layer, and progressively lower in the granular layer, deep nuclei and white matter. Different results have been obtained by quantitative autoradiography by Unnerstall et al. (1981) and Bowery et al. (1987), using as ligand [3H] muscimol or [3H] GABA (in the presence of unlabelled baclofen): in these studies the granule cell layer showed one of the highest levels of binding in the CNS, whereas moderate or low concentrations were found in the molecular layer. Fewer GABA A sites have been observed in deep nuclei (Bowery et al., 1987). Electronmicroscopical observations (Chan-Palay and Palay, 1978) have shown that the [3H] muscimol binding sites, presumably corresponding to GABA a receptors, are localized on the cell membrane of Purkinje, granule, basket and Golgi cells: the label is distributed in correspondence with synapses, which are axodendritic (89%) or axosomatic (11%; ChanPalay and Palay, 1978). Note that Somogyi et al. (1989) have reported that GABA A receptor immunoreactivity occurs both in the synaptic clefts and in the non-junctional areas of the membrane of GABA receptive cells. Immunoreactivity for GABAA receptors has been demonstrated in both Purkinje and granule cell layers also by Taguchi et al. (1989): Purkinje cell somata are surrounded by staining for GABAA receptors. Interestingly, a very high concentration of GABAB binding sites occurred in the molecular layer (higher than the density in GABAA sites). Moderate levels of GABA~ sites are found in the granular layer (Bowery et al., 1987). 7.9.2. G A B A e r g i c synaptic potentials in the cerebellar cortex

Cerebellar neurophysiology has been extensively reviewed by Eccles et al. (1967) and by Llin~s (1981); in addition, the role of GABA and other transmitters in cerebellar circuits was reviewed by Krnjevi6 (1982). The occurrence of graded, long-lasting hyperpolarizing potentials in Purkinje cells in response to parallel fibre activation was first described in the cat in vivo by Andersen et al. (1964c). Following high intensities of surface stimulation, this IPSP was preceded by a small EPSP and was present in all Purkinje cells within and at each side of the stimulated beam of parallel fibres. Andersen and co-workers argued that the most likely candidates for the generation of the IPSP are the basket cells through their synapses on the Purkinje cell body. The work of Eccles, Llimis and Sasaki (reviewed by Llin~s, 1981) has shown that the IPSP reverses with membrane hyperpolarization or

with intracellular C1- loading. Field potential data obtained by the same authors suggest a role by stellate cells in the inhibition of Purkinje cells. The pharmacology of Purkinje cell synaptic inhibition has been investigated with a variety of extracellular techniques. The IPSP produces a decrease in spontaneous or homocysteate-evoked firing of Purkinje cells. This phenomenon is abolished by iontophoretic or i.v. administration of bicuculline (Curtis et al., 1970a; Curtis and Felix, 1971) and is prolonged by i.v. diazepam (Curtis et al., 1976a), chlordiazepoxide or the benzodiazepine, RU 32007: the effect of this last compound has been shown to be antagonized by flumazenil (Gardner, 1986). Another extracellular correlate of the Purkinje cell IPSP, namely the field potential evoked by parallel fibre stimulation, has been shown to be insensitive to strychnine (Andersen et al., 1963b). Conditioning stimulation of parallel or mossy fibres inhibits the N~ field potential in the granule cell layer, which is generated by antidromic invasion of the Purkinje cells following juxtafastigial stimulation: this paired-pulse inhibition is an index of basket cell inhibitory action and is also antagonized by i.v. picrotoxin or bicuculline (Bisti et al., 1971). As far as granule cells are concerned, their activity is controlled by inhibition from Golgi cells: this synaptic process is readily demonstrated by measuring the inhibition of granule cell axonal discharges by parallel or mossy fibres. Golgi cell-mediated inhibition is greatly reduced or suppressed by i.v. bicuculline or picrotoxin (Bisti et al., 1971). 7.9.3. G A B A e r g i c synaptic potentials in deep cerebellar nuclei

The short latency monosynaptic IPSP evoked in Deiters' neurones by Purkinje cell activation was first described in the cat in vivo (Ito and Yoshida, 1966; Obata et al., 1967). The IPSP is graded and acquires a biphasic time course at the highest intensities of cerebellar surface stimulation (Ito and Yoshida, 1966). The GABAergic nature of this IPSP has been demonstrated by Obata et al. (1967, 1970), who showed that the synaptic potential has the same reversal potential as the Deiters' neurone response to GABA. Most pharmacological investigations have focussed on the extracellular correlates of the IPSP in deep cerebellar nuclei. The depression of antidromic invasion of Deiters' neurones induced by cerebellar surface stimulation (judged by the size of the associated field potential) was thus found to be sensitive to i.v. picrotoxin (Obata et al., 1970) and to i.v. or iontophoretic bicuculline (Curtis et al., 1970c). Similarly, iontophoretic or systemic administration of picrotoxin or bicuculline blocks the Purkinje cell-mediated depression of the spontaneous (or homocysteate-induced) firing of Deiters' (or nucleus interpositus) neurones (Obata et al., 1970; Kawaguchi and Ono, 1973). While Kawaguchi and Ono (1973) have reported that strychnine is not effective as an antagonist of the inhibition of cat interpositus neurones, contrary evidence has been obtained in tissue culture preparations by Wojtowicz et al. (1980).

GABA RECEPTORMECHANISMS Stimulation of the spinal cord produces in most of Deiters' neurones a complex synaptic response, comprising an early and a late EPSP-IPSP sequence, which is followed by a rebound excitability increase (Bruggencate et al., 1972a). IPSPs reverse with hyperpolarizing current injection and are probably generated by activation of Purkinje cells (Bruggencate et al., 1972a; Llinds, 1981). The subsequent increased excitability is due to the activation of inhibitory circuits in the cerebellar cortex, which in turn reduce Purkinje cell firing, causing disinhibition in Deiters' nucleus (Bruggencate et al., 1972a; Llin~s, 1981). 7.10. BRAINSTEMNUCLEI

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slow hyperpolarization is noradrenergic (Egan et al., 1983). In the presence of the excitatory amino acid antagonist kynurenic acid (500pM), the residual synaptic potential is abolished by 10 pM bicuculline and has a reversal potential similar to that of neuronal responses to bath-applied GABA ( - 7 1 vs - 7 8 mV in records obtained with K acetate-filled microelectrodes). Osmanovi6 and Shefner (1988, 1990) have shown that bath-applied GABA (in the presence of bicuculline) or baclofen can activate GABAB-receptor mediated mechanisms in locus coeruleus neurones in vitro. The role---if a n y - - o f these GABA8 sites in mediating synaptic responses remains to be investigated.

7.10.1. Dorsal raphe

Intense staining for GABA-like immunoreactivity is present in the neuropil of this nucleus (Ottersen and Storm-Mathisen, 1984). Although GABA-positive neurones appear to be scarce (Ottersen and StormMathisen, 1984), the presence of intrinsic cells positive for GAD or newly-synthesized GABA-T has been demonstrated by Nanopoulos et al. (1982) and Nagai et al. (1983). Moderate levels of GABAA and GABAB sites are also present (Bowery et al., 1987). Spontaneous GABAergic potentials (2-20mV in amplitude) blocked by 3 0 / t i bicuculline occur in approximately 30% of rat dorsal raphe neurones in vitro (Pan and Williams, 1989). In the same rat brain slice, stimulation of the area dorsal to the raphe nucleus evokes in raphe neurones an EPSP-IPSP sequence: the IPSP component, studied in the presence of excitatory amino acid antagonists, has the properties of a GABA A synaptic response, as it lasts up to 100 ms and is blocked by 30 tiM bicuculline. Furthermore, its reversal potential ( - 65 mV) is depolarized by intracellular CI- loading (Pan and Williams, 1989). 7.10.2. Locus coeruleus In this brainstem area, GAD is present in nerve terminals which synapse on the dendrites and cell bodies of noradrenergic neurones (Berod et al., 1984). Intrinsic locus coeruleus neurones do not display GAD immunoreactivity (Berod et al., 1984). Both GABAA and GABAB binding sites have been demonstrated in this area by autoradiography (Palacios et al., 1981). Ennis and Aston-Jones (1989) have shown that a significant component of the GABAergic afferents to the locus coeruleus originate from the nucleus prepositus hypoglossi: in fact, the depression of single unit firing induced in the locus coeruleus by stimulating the nucleus prepositus hypoglossi is sensitive to GABA antagonists. The properties of GABAergic synaptic potentials have been investigated in a rat locus eoeruleus slice preparation by Cherubini et al. (1988). Local stimulation evokes a triphasic depolarizinghyperpolarizing potential in these neurones: the early depolarization results from co-activation of excitatory amino acid receptors (predominantly of nonNMDA type) and of GABAA receptors, whereas the

7.11. SPINAL CORD 7.11.1. G A B A e r g i c pathways

GAD-immunoreactive puncta have been described in most laminae of the spinal grey matter. The intensity of neuropil staining is particularly heavy in Rexed laminae I-III and decreases in more ventral regions. Moderate levels are observed in layers IV-VI, in the medial aspect of the intermediate grey (lamina VII) and in lamina X, while a lower density of puncta is present in the ventral horn (McLaughlin et al., 1975). In the dorsal horn, these GABAergic terminals synapse on neuronal dendrites and cell bodies and, particularly in substantia gelatinosa (laminae II-III), on other axon terminals (McLaughlin et al., 1975). In the substantia gelatinosa, axon terminals postsynaptic to GAD-positive boutons have been identified as primary afferent terminals on the basis of their degeneration following dorsal rhizotomy (Barber et al., 1978). The same authors have also studied with electron microscopy the synaptic formations between the GABAergic terminals, the primary afferent terminals and the dendritic profiles of spinal neurones: the types of synaptic rosettes thus described include simple arrangements between one GABAergic terminal and one primary afferent terminal or more complex arrangements in which the GABAergic bouton establishes two synaptic contacts, one on the primary afferent terminal and the other on the same dendritic profile which receives the afferent terminal. Such a structure is termed "triadic" and has been discussed by Fyffe and Light (1984). In the ventral horn, GABAergic boutons form synaptic contacts on large and small dendrites of motoneuronal cell bodies and on larger axon terminals which are presynaptic to motoneuronal somata (McLaughlin et al., 1975). Cell bodies stained for GAD-immunoreactivity or for [3H] GABA uptake are small or medium size and are seen mostly within outer lamina II and at the border between laminae II and III (Barber et al., 1982; Hunt, 1983). Very low levels of GABAA binding sites are found throughout the spinal cord, with the exception of the intermediate layer of lamina X, which has low levels. Moderate levels of GABAB receptors are present in the substantia gelatinosa and low levels in lamina X (Bowery et al., 1987).

78

L. SIVILOTT1and A. NISTRX 7. l 1.2. G A B A e r g i c synaptic potentials

Recurrent inhibition in spinal cord motoneurones was first described by Renshaw (1941) and extensively investigated by Eccles and co-workers (for a review see Eccles, 1964). Antidromic stimulation of the axon of a motoneurone evokes a hyperpolarization of the motoneuronal membrane, regardless of whether a full antidromic action potential has been elicited. The hyperpolarization has the characteristics of a disynaptic IPSP and is produced by motor axon collaterals activating inhibitory interneurones in the spinal cord (Renshaw cells). While the depression by systemic strychnine of recurrent inhibition (Eccles et al., 1954) argues for a glycinergic mediation of this phenomenon (Werman et al., 1968), there is evidence that GABA may also be involved. In fact, Larson (1969) has found that the later phase of this IPSP is less sensitive to strychnine than the earlier phase. Furthermore, Larson and Major (1970) have reported that hexobarbitone prolongs the recurrent IPSP. Stronger evidence comes from the work by Cullheim and Kellerth (1981): these authors found that in the pentobarbitone-anaesthetized cat i.v. strychnine or bicuculline selectively antagonize the early or the late phase of the recurrent IPSP, respectively. Simultaneous administration of strychnine and bicuculline entirely suppresses the IPSP and its extracellular equivalent, namely the ventral root-induced depression of the monosynaptic reflex. The latter inhibitory phenomenon is also abolished by i.v. administration of strychnine and picrotoxin. Polc and Haefely (1982) have shown that in spinalized, unanaesthetized cats the late component of the recurrent inhibition of monosynaptic ventral root reflexes is enhanced by i.v. diazepam or midazolam. The same authors have also confirmed that this component is depressed by bieuculline, but not by strychnine. Other extracellular studies have however found that recurrent inhibition of spinal motoneurones is completely abolished by systemic strychnine and is not affected by bicuculline (Curtis et al., 1976b). While it is difficult to speculate on the reasons for this discrepancy, Cullheim and Kellerth (1981) have suggested that the parameters of stimulation chosen for both the conditioning and the test shocks may influence the pharmacology of the observed inhibition. In fact, in their experiments no strychnine-resistant GABAergic component could be detected when m a x i m a l intensities of stimulation were used as test shocks to elicit the monosynaptic reflex. Furthermore, their intracellular results show that the presence or absence of strychnine-insensitive IPSP components depends, in the same motoneurone, upon the motor nerve chosen for stimulation. No pattern, based on motor pool category or on motor unit type, could however be detected. In 1957 Frank and Fuortes reported that conditioning shocks applied to muscle afferents reduce the size of monosynaptic EPSPs elicited in spinal motoneurones by stimulation of another muscle nerve. This phenomenon has been extensively investigated, particularly by Eccles and co-workers (for reviews see Eccles, 1964; Burke and Rudomin, 1977; Nistri, 1983; Davidoff and Hackman, 1985). Inhibition of EPSPs

evoked by Ia, Ib or cutaneous afferents can be produced by trains of stimuli applied to afferent fibre groups. The depression of EPSP amplitude is fairly modest, typically by about one third of the control amplitude after a short train of conditioning stimuli. The peak inhibition is observed 10-20ms after a conditioning volley of four shocks, but the EPSP does not recover its control amplitude for at least 100 ms. With conventional intracellular recordings from spinal motoneurones no postsynaptic potential or change in neuronal excitability is usually detected in response to the conditioning stimulation (Davidoff and Hackman, 1985). This finding suggests that this type of inhibition is either produced presynaptically (on the afferents that mediate the EPSP), or generated postsynaptically (on dendritic sites remote from the motoneuronal soma from which intracellular records are obtained; Frank, 1959). There is now considerable evidence to support the role of presynaptic mechanisms in most (if not all) of the EPSP depression described above. Indeed, the inhibition of EPSPs is associated with a slow depolarization of afferent fibres (primary afferent depolarization or PAD), which can be recorded as a dorsal root potential from the root used for the test volleys or from adjacent roots. The time course of the inhibition of EPSPs (and, consequently, of monosynaptic reflex discharges) is similar to that of PAD recorded intracellularly (from Ia group afferent terminals) or extraeellularly (Eccles, 1964; Davidoff and Hackman, 1985). The PAD is accompanied by an increase in the conductance of the afferent fibre terminal (Padjen and Hashiguchi, 1983). The inhibition of the EPSP is also correlated with an increase in primary afferent excitability, as tested by applying current pulses to the terminal region of these fibres. There is also strong albeit indirect evidence that presynaptic inhibition is mediated by GABA acting on primary afferent terminals: indeed G A B A depolarizes primary afferents and increases their excitability. Furthermore, both the response to GABA and PAD show the properties of GABAA-mediated phenomena, since they are antagonized by picrotoxin or bicuculline and enhanced by barbiturates or benzodiazepines (Curtis et al., 1971; Nicoll et al., 1975; Barker et al., 1975a,b; Polc, 1988). Technical difficulties preclude detailed investigations into the pharmacology of PAD with intracellular, actually intrafibre recording, hence isolated preparations of dorsal root ganglia, which contain the somata of primary afferents, have been used as a model. Work carried out in dorsal root ganglia in vitro by Feltz and colleagues (D6sarmenien et al., 1984; Schlichter et al., 1987) has demonstrated the presence of both GABAA and GABAB receptors on all categories of A,~ and C primary afferent neurones. Edwards et al. (1989) have recently shown that i.v. baclofen reduces Ia monosynaptic EPSPs by a presynaptic mechanism of action in the cat in vivo. This finding suggests that GABAB receptors might also be present on group Ia afferents, which are traditionally regarded as possessing GABAA receptors (Nistri, 1983). The extent to which GABA a receptor activation contributes to presynaptic inhibition evoked by conditioning stimulation of spinal afferents remains, however, to be clarified.

GABA RECEPTORMECHANISMS It must be pointed out that the phenomenon of presynaptic inhibition and of P A D might be due in part to an increase in the K + concentration of the extracellular medium around primary afferent terminals. In particular, a contribution by this K ÷ accumulation to the genesis of the late part of P A D (which is insensitive to GABAergic antagonists) is more likely when conditioning is produced by prolonged, high frequency stimulation (Davidoff et al., 1988). Finally, C o o k and Cangiano (1972) and Carlen et al. (1980) have reported that minimal conditioning stimuli normally associated with " p u r e " presynaptic inhibition, do produce small, long-lasting IPSPs in spinal motoneurones. Conditioning volleys have also been shown by short pulse analysis to produce an increase in the motoneuronal conductance (Carlen et al., 1980). A small contribution by remote postsynaptic inhibitory mechanisms, possibly GABAergic, cannot thus be excluded on electrophysiological grounds and is made possible by the histological observation of "triadic" GABAergic synapses comprising pre- and postsynaptic elements. Acknowledgements--We are grateful to Miss Carol Brown for invaluable assistance in the preparation of the manuscript. L. S. was supported by the Joint Research Board of St. Bartholomew's Hospital.

REFERENCES ABEL, M. S., BLUME, A. J. and GARRETT, K. M. (1989) Differential effects of iodide and chloride on allosteric interactions of the GABA A receptor. J. Neurochem. 53, 940-945. AGEY, M. W. and DUNN, S. M. J. (1989) Kinetics of [3H]muscimol binding to the GABAA receptor in bovine brain membranes. Biochemistry 28, 4200-4208. AICKIN, C. C. and DEISZ, R. A. (1981) Pentobarbitone interference with inhibitory synaptic transmission in crayfish stretch receptor neurones. J. Physiol., Lond. 315, 175-187. AKAIKE,N., HATTORI,K., OOMURA,Y. and CARPENTER,D. (1985) Bicuculline and picrotoxin block y-aminobutyric acid-gated C1- conductance by different mechanisms. Experientia 41, 70-71. ALGER, B. E. (1984) Characteristics of a slow hyperpolarizing synaptic potential in rat hippocampal pyramidal cells in vitro. J. Neurophysiol. 52, 892-910. ALGER, B. E. (1985) GABA and glycine: postsynaptic actions. In: Neurotransmitter ,4ctions in the Vertebrate Nervous System, pp. 33-69. Eds. M. A. ROGAWSKYand J. L. BARKER.Plenum Press: New York. ALGER, B. E. and NICOLL, R. A. (1980a) Spontaneous inhibitory post-synaptic potentials in hippocampus: mechanism for tonic inhibition. Brain Res. 200, 195-200. ALGER, B. E. and NICOLL, R. A. (1980b) The epileptiform burst after-hyperpolarization: A calcium-dependent potassium potential in hippocampal pyramidal cells. Science 210, 1122-1124. ALGER, B. E. and NICOLL, R. A. (1982a) Pharmacological evidence for two kinds of GABA receptor on rat hippocampal pyramidal cells studied in vitro. J. Physiol., Lond. 328, 125-141. ALGER, B. E. and NICOLL, R. A. (1982b) Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro. J. Physiol., Lond. 328, 105-123. ALLEN, G. I., ECCLES,J. C., NICOLL, R. A., OSHIMA,T. and RUmA, F. J. (1977) The ionic mechanisms concerned in JPN36/I--F

79

generating the i.p.s.p, of hippocampal pyramidal cells. Proc. R. Soc. Lond. B. 198, 363-384. ALLERTON, C. A., BODEN, P. R. and HILL, R. G. (1989) Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro. Br. J. Pharmac. 96, 29-38. ANDERSEN, P., ECCLES, J. C. and LefYNING, Y. (1963a) Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature 198, 540-542. ANDERSEN,P., ECCLES,J. C., L,efYNING,Y. and VOORHOEVE, P. E. (1963b) Strychnine-resistant central inhibition. Nature 200, 843-845. ANDERSEN, P., ECCLES, J. C. and LO~YNING,Y. (1964a) Location of postsynaptic inhibitory synapses on hippocampal pyramids. J. Neurophysiol. 27, 592-607. ANDERSEN, P., ECCLES, J. C. and L~YNING, Y. (1964b) Pathway of postsynaptic inhibition in the hippocampus. J. NeurophysioL 27, 608-619. ANDERSEN,P., ECCLES,J. C. and VOORHOEVE,P. E. (1964c) Postsynaptic inhibition of cerebellar Purkinje ceils. J. Neurophysiol. 27, 1138-1153. ANDERSEN,P., ECCLES,J. C. and SEARS,T. A. (1964d) The ventro-basal complex of the thalamus: types of cells, their responses and their functional organization. J. Physiol., Lond. 174, 370-399. ANDERSEN,P., HOLMQVIST,B. and VOOgnOEVE,P. E. (1966) Entorhinal activation of dentate granule cells. Acts physiol. scand. 66, 448-460. ANDERSEN,P., DINGLEDINE,R., GJERSTAD,L., LANGMOEN,I. A. and MOSFELDLAURSEN,A. (1980) Two different responses of hippocampal pyramidal ceils to application of gamma-aminobutyric acid. J. Physiol., Lond. 305, 279-296. ANDERSON,C. R. and STEVENS,C. F. (1973) Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction. J. Physiol., Lond. 235, 655-691. ANDRADE, R., MALENKA,R. C. and NICOLL, R. A. (1986) A G protein couples serotonin and GABA B receptors to the same channels in hippocampus. Science 234, 1261-1265. ANHOLT, R. R. H., DESOUZA,E. B., OSTER-GRANITE,M. L. and SNYDER,S. H. (1985) Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. J. Pharmac. exp. Ther. 233, 5t7-526. ARAKAWA,Z. and OKADA,Y. (1988) Excitatory and inhibitory action of GABA on synaptic transmission in the guinea pig superior colliculus slices. Eur. J. Pharmac. 158, 217-224. ASHWOOD, T. J., COLLINGRIDGE,G. L., HERRON, C. E. and WI-IEAL, H. V. (1987) Voltage-clamp analysis of somatic ~,-aminobutyric acid responses in adult rat hippocampal CAI neurones in vitro. J. Physiol., Lond. 384, 27-37. AUERBACH,A., DEL CASTILLO,J., SPECHT,P. C. and TITMUS, M. (t983) Correlation of agonist structure with acetylcholine receptor kinetics: studies on the frog end-plate and on chick embryo muscle. J. Physiol., Lond. 343, 551-568.

AVOLI, M. (1986) Inhibitory potentials in neurons of the deep layers of the in vitro neocortical slice. Brain Res. 370, 165-170. AVOLI, M. and PERREAULT,P. (1987) GABAergic depolarizing potential in the hippocampus disclosed by the convulsant 4-aminopyridine. Brain Res. 400, 191-195. BARBACCIA, M. L., COSTA, E. and GUIDOTTI, A. (1988) Endogenous ligands for high-affinity recognition sites of psychotropic drugs..4. Rev. Pharmac. Toxicol. 28, 451-476. BARBER,R. P., VAUGHN,J. E., SAITO,K., McLAUGHLIN,B. J. and ROBERTS, E. (1978) GABAergic terminals are

80

L. SIVILOTTI and A. NISTRI

presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord. Brain Res. 141, 35-55. BARBER, R. P., VAUGHN, J. E. and ROBERTS, E. (1982) The cytoarchitecture of GABAergic neurons in rat spinal cord. Brain Res. 238, 305-328. BARKER, J. L. and HARRISON, N. L. (1988) Outward rectification of inhibitory postsynaptic currents in cultured rat hippocampal neurones. J. Physiol., Lond. 403, 41-56. BARKER, J. L. and MATHERS, D. A. (1981) GABA analogues activate channels of different duration on cultured mouse spinal neurons. Science 212, 358-361. BARKER,J. L. and McBURNEY, R. N. (1979) Phenobarbitone modulation of postsynaptic GABA receptor function on cultured mammalian neurons. Proc. R. Soc. Lond. B. 206, 319-327. BARKER~ J. L. and RANSOM, B. R. (1978) Amino acid pharmacology of mammalian central neurones grown in tissue culture, d. Physiol., Lond. 280, 331-354. BARKER, J. L., NICOLL, R. A. and PADJEN, A. (1975a) Studies on convulsants in the isolated frog spinal cord. I. Antagonism of amino acid responses. J. Physiol., Lond. 245, 521 536. BARKER, J. L., NICOLL, R. A. and PADJEN, m. (1975b) Studies on convulsants in the isolated frog spinal cord. II. Effects on root potentials. J. Physiol., Lond. 245, 537-548. BARKER, J. L., McBURNEY, R. N. and MACDONALD, J. F. (1982) Fluctuation analysis of neutral amino acid responses in cultured mouse spinal neurones. J. Physiol., Lond. 322, 365-387. BARKER, J. L., McBURNEY, R. N. and MATHERS, n . A. (1983) Convulsant-induced depression of amino-acid responses in cultured mouse spinal neurones studied under voltage clamp. Br. J. Pharmac. 80, 619-629. BARKER, J. L., HARRISON, N. L., LANGE, G. D. and OWEN, D. G. (1987) Potentiation of 7-aminobutyric-acid-actirated chloride conductance by a steroid anaesthetic in cultured rat spinal neurones. J. Physiol., Lond. 386, 485-501. BARNARD, E. A., DARLISON, M. G. and SEEBURG,P. (1987) Molecular biology of the GABA A receptor. The receptor/channel superfamily. Trends Neurosci. 10, 502-509. BEATTIE, D. T., CURTIS, D. R., DEBAERT, M., VACCHER, C. and BERTHELOT, P. (1989) Baclofen antagonism by 4amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid in the cat spinal cord. Neurosci. Lett. 100, 292-294. BEN-ARI, Y., KRNJEVI6, K. and REINHARDT,W. (1979) Hippocampal seizures and failure of inhibition. Can. J. Physiol. Pharmac. 57, 1462-1466. BEN-Am, Y., KRNJEVI(~, K., REIFFENSTEIN, R, J. and REINHARDT, W. (1981) Inhibitory conductance changes and action of )'-aminobutyrate in rat hippocampus. Neuroscience 6, 2445-2463. BEN-ARI, Y., CHERUBINI, E., CORRADETTI, R. and GAJARSA, J.-L. (1989) Giant synaptic potentials in immature rat CA3 hippocampal neurones. J. Physiol., Lond. 416, 303 325. BERNARDI, G., MARCIANI, U. G., MOROCUTTI, C. and GIACOMINI, P. (1976) The action of picrotoxin and bicuculline on rat caudate neurons inhibited by GABA. Brain Res. 102, 379-384. BEROO, A., CHAT, M., PONT, L. and TAPPAZ, M. (1984) Catecholaminergic and GABAergic anatomical relationship in the rat substantia nigra, locus coeruleus, and hypothalamic median eminence: immunocytochemical visualization of biosynthetic enzymes on serial semithin plastic embedded sections. J. Histochem. Cytochem. 32, 1331-1338. BIEDENBACH, M. A. and STEVENS, C. F. (1969) Synaptic organization of cat olfactory cortex as revealed by intracellular recording. J. Neurophysiol. 32, 204-214. BISCOE, T. J. and DUCnEN, M. R. (1985a) The anion selectivity of GABA-mediated post-synaptic potentials in mouse hippocampal cells. Q. JI exp. Physiol. 70, 305-312.

BISCOE, T. J. and DUCI-IEN, M. R. (1985b) Actions and interactions of GABA and benzodiazepines in the mouse hippocampal slice. Q. Jl exp. Physiol. 70, 313-328. BISCOE, T. J. and DUCHEN, M. R. (1985c) An intracellular study of dentate, CA1 and CA3 neurones in the mouse hippocampal slice. Q. Jl exp. Physiol. 70, 189-202. BISCOE, T. J. and STRAUGHAN,D. W. (1966) Micro-electrophoretic studies of neurons in the cat hippocampus. J. Physiol., Lond. 183, 341-359. BISTI, S., IOSIF, G., MARCHESI, G. F. and STRATA,P. (1971) Pharmacological properties of inhibitions in the cerebellar cortex. Expl Brain Res. 14, 24-37. BLAIR, L. A. C., LEVITAN, E. S., MARSHALL, J., DIONNE, V. E. and BARNARD, E. A. (1988) Single subunits of the GABA A receptor form ion channels with properties of the native receptor. Science 242, 577-579. BOLAM,J. P., CLARKE,D. J., SMITH,A. D. and SOMOGYI,P. (1983) A type of aspiny neuron in the rat neostriatum accumulates [DH] ?-aminobutyric acid: combination of Golgi-staining, autoradiography, and electron microscopy. J. comp. Neurol. 213, 121-134. BONANNO, G., PELLEGRINI,G., ASARO, D., FONTANA,G. and RAITERI, M. (1989a) GABA a autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions. Eur. J. Pharm. molec. Pharmac. 172, 41-49. BONANNO, G., CAVAZZANI,P., ANDRIOLI, G. C., ASARO, D., PELLEGRINI,G. and RAITERI, M. (1989b). Release-regulating autoreceptors of the GABAB-type in human cerebral cortex. Br. J. Pharmac. 96, 341-346. BORMANN, J. (1988) Electrophysiology of GABA A and GABA B receptor subtypes. Trends Neurosci. 11, 112-116. BORMANN, J. and CLAPHAM, D. E. (1985) 7-Aminobutyric acid receptor channels in adrenal chromaffin cells: A patch clamp study. Proc. HatH. Acad. Sci. U.S.A. 82, 2168-2178. BORMANN, J. and KETTENMANN, H. (1988) Patch-clamp study of 7-aminobutyric acid receptor Cl- channels in cultured astrocytes. Proc. HatH. Acad. Sci. U.S.A. 85, 9336-9340. BORMANN, J., HAMILL, O. P. and SAKMANN, B. (1987) Mechanisms of anion permeation through channels gated by glycine and ~,-aminobutyric acid in mouse cultured spinal neurones. J. Physiol., Lond. 385, 243-286. BOWERY, N. G. (1982) Baclofen: 10 years on. Trends Pharmac. Sci. 3, 400-403. BOWERY, N. G. (1989) GABA B receptors and their significance in mammalian pharmacology. Trends Pharmac. Sci. I0, 401-407. BOWERY, N. G., HILL, D. R. and HUDSON, A. L. (1983) Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. Br. J. Pharmae. 78,

191-206. BOWERY, N. G., HUDSON, A. L. and PRICE, G. W. (1987) GABAA and GABAB receptor site distribution in the rat central nervous system. Neuroscience 20, 365-383. BOWLING, A. C. and DELORENZO, R. J, (1982) Micromolar affinity benzodiazepine receptors: identification and characterization in central nervous system. Science 216, 1247-1250. BRAESTRUP, C. and NIELSEN, M. (1981) GABA reduces binding of [DH]-methyl fl-carboline-3-carboxylate to brain benzodiazepine receptors. Nature 294, 472-474. BRAESTRUP, C., NIELSEN,M., KROGSGAARD-LARSEN,P. and FALCH, E. (1979) Partial agonists for brain GABA/benzodiazepine receptor complex. Nature 280, 331-333. BRANCH, C. L. and MARTIN, A. R. (1958) Inhibition of Betz cell activity by thalamic and cortical stimulation. J. Neurophysiol. 21, 380-390. BRENNAN, M. J. W., CANTRILL, R. C., OLDFIELD, M. and KROGSGAARD-LARSEN,P. (I 98 I) Inhibition of 7-aminobu-


81

tyric acid release by ),-aminobutyric acid agonist drugs. (1984). Inhibitory post-synaptic currents in rat hippocamPharmacology of the y-aminobutyric acid autoreceptor. pal CA1 neurones. J. Physiol., Lond. 356, 551-564. Molec. Pharmac. 19, 27-30. COLLINS, G. G. S., ANSON, J. and KELLY, E. P. (1982). BRUGGENCATE, G. TEN, TEICHMANN, R. and WELLER, E. Baclofen: Effects on evoked field potentials and amino (1972a) Neuronal activity in the lateral vestibular nucleus acid neurotransmitter release in rat olfactory cortex slice. of the cat. I. Patterns of postsynaptic potentials and Brain Res. 238, 371-383. discharges in Deiters neurones evoked by stimulation of COLQUHOUN, D. (1981) How fast do drugs work? In: the spinal cord. Pfliigers Jtrch. 337, 119-134. Towards Understanding Receptors. Current Reviews in BRUGGENCATE, C. TEN, TEICHMANN, R. and WELLER, E. Biomedicine L PP. 16-27. Ed. J. W. LAMBLE. Elsevier/ (1972b) Neuronal activity in the lateral vestibular nucleus North Holland: Amsterdam. of the cat. Ill. Inhibitory actions of cerebellar Purkinje COLQUHOUN, D. and SAKMANN, B. (1985) Fast events in cells evoked via mossy and climbing fibre afferents. single-channel currents activated by acetycholine and its Pfliigers Arch. 337, 147-162. analogues at the frog muscle endplate. J. Physiol., Lond. BUCHWALD, N. A., PRICE, D. D., VERNON, L. and HULL, C. 369, 501-557. D. (1973) Caudate intracellular response to thalamic and COLQtmOUN, D., DIONNE, V. E., STEINBACH, J. H. and cortical inputs. Expl NeuroL 38, 311-323. STEVENS,C. F. (1975) Conductance of channels opened by BURKE, R. E. and RUDOMIN, P. (1977) Spinal neurons and acetycholine-like drugs in muscle end-plate. Nature 253, synapses. In: Handbook of Physiology, Section 1: The 204-206. Nervous System Vol. 1, Cellular Biology of Neurons Part CONNORS, B. W., GUTNICK,M. J. and PRINCE,D. A. (1982) 2, pp. 877-944. Ed. E. R. KANDEL. American PhysiologiElectrophysiological properties of neocortical neurons in vitro. J. Neurophysiol. 4g, 1302-1320. cal Society: Bethesda. BURKE,W. and SEFTON,A. J. (1966) Inhibitory mechanisms CONNORS, B. W., MALENKA, R. C. and SILVA, L. R. (1988) in lateral geniculate nucleus of rat. J. Physiol., Lond. 187, Two inhibitory postsynaptic potentials and GABA A and 231-246. GABA s receptor-mediated responses in neocortex of rat BUZSA,KI, G. (1984) Feed-forward inhibition in the hippoand cat. J. Physiol., Lond. 406, 443~,68. campal formation. Prog. Neurobiol. 22, 131-153. CooK, W. R. JR and CANGIANO, A. (1972) Presynaptic and CARLEN, P. L., WERMAN, R. and YAARI, Y. (1980) Postpostsynaptic inhibition of spinal motoneurones. J. Neurosynaptic conductance ini:rease associated with presynphysiol. 35, 389-403. aptic inhibition in cat lumbar motoneurones. J. Physiol., COOMaS, J. S., ECCLES, J. C. and FATT, P. (1955) The specific Lond. 298, 539-557. ionic conductances and the ionic movements across the CASALOTTI, S. O., STEPHENSON, F. A. and BARNARD, E. A. motoneuronal membrane that produce the inhibitory (1986) Separate subunits for agonists and benzodiazepine post-synaptic potential. J. Physiol. 130, 326-373. binding in the y-aminobutyric acidA receptor oligomer. J. CORRADETTI, R., RUGGIERO, M., CHIARUGI, V. P. and biol. Chem. 261, 15013-15016. PEPEU, G. (1987) GABA-receptor stimulation enhances CASH, D. J. and SUBBARAO,K. (1987a) Desensitization of norepinephrine-induced polyphosphoinositide metab7-aminobutyric acid receptor from rat brain: two disolism in rat hippocampal slices. Brain Res. 411, 196-199. tinguishable receptors on the same membrane. BiochemCOSTA, T., ROOBARD, D. and PERT, C. B. (1979) Is the istry 26, 7556-7562. benzodiazepine receptor coupled to a chloride anion CASH, D. J. and SUBBARAO,K. (1987b) Channel opening of channel? Nature 277, 315-317. 7-aminobutyric acid receptor from rat brain: molecular COTTRELL, G. A., LAMBERT, J. J. and PETERS, J. A. (1987) mechanisms of the receptor responses. Biochemistry 26, Modulation of GABA A receptor activity by alphaxalone. 7562-7570. Br. J. Pharmac. 90, 491-500. CHAN-PALAY, V. (1978) Autoradiographic localization of CRUNELLI, V., HABY, M., JASSIK-GERSCHENFELD, D., 7-aminobutyric acid receptors in the rat central nervous LEP,ESCrm, N. and Pmcalo, M. (1988) CI- and K+-depen system by using [3H]muscimol. Proc. natn. Acad. Sci. dent inhibitory postsynaptic potentials evoked by interU.S.A. 75, 1024-1028. neurones of the rat lateral geniculate nucleus. J. PhysioL, CHAN-PALAY, V. and PALAY, S. t . (1978) Ultrastructural Lond, 399, 153-176. localization of y-aminobutyric acid receptors in the CULL-CANDY. S. G. and OGDEN, D. C. (1985) Ion channels mammalian central nervous system by means of activated by L-glutamate and GABA in cultured cerebel[3H]muscimol binding. Proc. natn. Acad. Sci. U.S.A. 75, lar neurones of the rat. Proc. R. Soc. Lond. B. 224, 2977-2980. 367-373. CHAN-PALAY, V., N1LAVER, G., PALAY, S. L., BEINFELD, CULL-CANnY,S. (J. and USOWlCZ, M. M. (1989) Whole-cell M. C., ZIMMERMANN,E. A., Wu, J.-Y. and O'DONOHUE, current noise produced by excitatory and inhibitory T. L. (1981 ) Chemical heterogeneity in cerebellar Purkinje amino acids in large cerebellar neurones of the rat. J. cells: Existence and co-existence of glutamic acid decarPhysiol., Lond. 415, 533-553. boxylase-like and motilin-like immunoreactivities. Proc. CULLHEIM, S. and KELLERTH, J. O. (1981) Two kinds of natn. Acad. Sci. U.S.A. 78, 7787-7791. recurrent inhibition of cat spinal g-motoneurones as CHEN, Q. X., STELZER, A., KAY, A. R. and WONG, R. K. S. differentiated pharmacologically. J. Physiol., Lond. 312, (1990) GABA A receptor function is regulated by phos209-224. phorylation in acutely dissociated guinea-pig hippocamCURTIS, D. R. and ECCLES, J. C. (1960) Synaptic action pal neurones. J. Physiol., Lond. 420, 207-221. during and after repetitive stimulation. J. Physiol., Lond. CHERUBINI, E., NORTH, R. A. and WILLIAMS,J. T. (1988) 150, 374-398. Synaptic potentials in rat locus coeruleus neurones, d. CURTIS, D. R. and FELIX, D. (1971) The effect of bicuculline Physiol., Lond. 406, 431-442. upon synaptic inhibition in the cerebral and cerebellar CHOW, P. and MATHERS, D. (1986) Convulsant doses cortices of the cat. Brain Res. 34, 301-321. of penicillin shorten the lifetime of GABA-induced chanCURTIS, D. R. and GYNTHER, B. D. (1986) Pitrazepin: a nels in cultured central neurones. Br. J. Pharmac. $$, central glycine and GABA antagonist. Eur. J. Pharmac. 541-547. 131, 311-313. COLLINGRIDGE, G. L. and DAVIES,J. (1981) The influence of CURTIS, D. R. and TEBECIS, A. K. (1972) Bicuculline and striatal stimulation and putative neurotransmitters on thalamic inhibition. Expl Brain Res. 16, 210-218. identified neurones in the rat substantia nigra. Brain Res. CURTIS, D. R., DUGGAN, A. W., FELIX, D. and JOHNSTON, 212, 345-359. G. A. R. (1970a) Bicuculline and central GABA recepCOLLINGRIDGE, G. L., GAGE, P. W. and ROBERTSON, B. tors. Nature 228, 676-677.

82

L. SIVILOTT1and A. NISTRI

CURTIS, D. R., FELIX, D. and MCLENNAN, H. (1970b) GABA and hippocampal inhibition. BE. J. Pharmac. 40, 881-883. CURTIS, D. R., DUGGAN, A. W. and FELIX, D. (1970c) GABA and inhibition of Deiters' neurones. Brain Res. 23, 117-120. CURTIS, D. R., DUGGAN, A. W., FELIX, D. and JOHNSTON, G. A. R. (1971) Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat. Brain Res. 32, 69-96. CURTIS, D. R., GAME, C. J. A., JOHNSTON, G. A. R. and MCCULLOCH, R. M. (1974) Central effects of fl-(pchlorophenyl)-~/-aminobutryic acid. Brain Res. 70, 493-499. CURTIS, D. R., LODGE, D., JOHNSTON, G. A. R. and BRAND, S. J. (1976a) Central actions of benzodiazepines. Brain Res. 118, 344-347. CURTIS, D. R., GAME, C. J. A., LODGE, D. and MCCULLOCH, R. M. (1976b) A pharmacological study of Renshaw cell inhibition. J. Physiol., Lond. 258, 227-242. DAVIDOFF, R. A. and HACKMAN, J. C. (1985) GABA: presynaptic actions. In: Neurotransmitter Actions in the Vertebrate Nervous System, pp. 3-32. Eds. M. A. ROGAWSKI and J. L. BARKER. Plenum Press: New York. DAVIDOFF, R. A. and SEARS, E. S. (1974) The effects of Lioresal on synaptic activity in the isolated spinal cord. Neurology 24, 957-963. DAVIDOFF, R. A., HACKMAN,J. C., HOLOHEAN,A. M., VEGA, J. U and ZHANG,D. X. (1988) Primary afferent activity, putative excitatory transmitters and extracellular potassium levels in frog spinal cord. J. Physiol., Lond. 397, 291-306. DAVIES, J. (1981) Selective depression of synaptic excitation in cat spinal neurones by baclofen: an iontophoretic study. BE. J. Pharmac. 72, 373-384. DAVIES, J. and WATKINS, J. C. (1974) The action of //phenyl-GABA derivatives on neurones of the cat cerebral cortex. Brain Res. 70, 50t-505. DEAN, P., REDGRAVE, P. and WESTBY, G. W. M. (1989) Event or emergency? Two response systems in the mammalian superior colliculus. Trends Neurosci. 12, 137-147. DEFRANCE, J. F., SHIMONO, T. and KITAI, S. T. (1972) Hippocampal inputs to the lateral septal nucleus: patterns of facilitation and inhibition. Brain Res. 37, 333-339. DEFRANCE, J. F., YOSHIrIARA, H., MCCREA, R. A. and KITAI, S. T. (1975) Pharmacology of the inhibition in the lateral septal region. Expl Neurol. 40, 502-523. DEFRANCE, J. F., YOSHIHARA, H. and CHRONISTER, R. B. (1976) Electrophysiological studies of the septal nuclei. I. The lateral septal region. Expl Neurol. 53, 399-419. DEFRANCE, J. F., MARCHAND, J. F., SirEs, R. W., CHRONISTER, R. B. and HUaaARD, J. I. (1985) Characterization of fimbria input to nucleus accumbens. J. Neurophysiol. 54, 1553-1567. DEISZ, R. A. and PRINCE, D. A. (1989) Frequency-dependent depression of inhibition in guinea-pig neocortex in vitro by GABA B receptor feedback on GABA release. J. Physiol., Lond. 412, 513-541. D~:SARMENIEN,M., FELTZ, P., OCCHIPINTI, G., SANTANGELO, F. and SCHLICHTER, R. (1984) Co-existence of GABA A and GABA B receptors on A6 and C primary afferents. BE. J. Pharmac. 81, 327-333. DIAMOND, J. M. and WRIGHT, E. M. (1969) Biological membranes: the physical basis of ion and non electrolyte selectivity. A. Rev. Physiol. 31, 581-646. DICHIARA, G., PORCEDDU, M. L., MORELLI, M., MULAS, M. L. and GESSA, G. L. (1979) Evidence for a GABAergic projection from the substantia nigra to the ventromedial thalamus and to the superior colliculus of the rat. Brain Res. 176, 275-284. DICHTER, M. A. and AYALA, G. F. (1987) Cellular mechanisms of epilepsy: a status report. Science 237, 157-164.

DINGLEDINE, R. and KORN, S. J. (1985)),-Aminobutyric acid uptake and the termination of inhibitory synaptic potentials in the rat hippocampal slice. J. Physiol., Lond. 366, 387-409. DINGLEDINE, R. and LANGMOEN, I. A. (1980) Conductance changes and inhibitory actions of hippocampal recurrent IPSPs. Brain Res. 185, 277-287. DOLPHIN, A. C. and SCOTT, R. H. (1986) Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen. BE. J. Pharmac. 88, 213-220. DOLPHIN, A. C. and SCOTT, R. H. (1987) Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides. J. Physiol., Lond. 386, 1-17. DOLPHIN, A. C., McGUIRK, S. M. and SCOTT, R. H. (1989) An investigation into the mechanisms of inhibition of calcium channel currents in cultured sensory neurones of the rat by guanine nucleotide analogues and ( - ) baclofen. BE. J. Pharmac. 97, 263-273. DREIFUSS, J. J., KELLY, J. S. and KRNJEVI~, K. (1969) Cortical inhibition and y-aminobutyric acid. Expl Brain Res. 9, 137-154. DREXLER, G. and SIEGHART,W. (1984) Evidence for association of a high affinity avermectin binding site with the benzodiazepine receptor. Eur. J. Pharmac. 101, 201-207. DUDEK, F. E., DEADWYLER, S. A., COTMAN,C. W. and LYNCH, G. (1976) Intracellular responses from granule cell layer in slices of rat hippocampus: perforant path synapse. J. NeurophysioL 39, 384-393. DUNLAP, K. (1981) Two types of y-aminobutyric acid receptors on embryonic sensory neurones. BE. J. Pharmac. 74, 579-585. DUNN, S. M. J., MARTIN, C. R., AGEY, M. W. and MIYAZAKI, R. (1989). Functional reconstitution of the bovine brain GABAA receptor from solubilized components. Biochemistry 28, 2545-2551. DUTAR, P. and NICOLL, R. A. (1988a) A physiological role for GABA B receptors in the central nervous system. Nature 332, 156-158. DUTAR, P. and NICOLL, R. A. (1988b) Pre- and postsynaptic GABAB receptors in the hippocampus have different pharmacological properties. Neuron 1, 585-591. DUTAR, P., LAMOUR, Y. and JOBERT, A. (1985) Septohippocampal neurones in the rat: an in vivo intracellular study. Brain Res. 340, 135-142. DWYER, T. M., ADAMS, D. J. and HILLE, B. (1980) The permeability of the endplate channel to organic cations in frog muscle. J. Ken. Physiol. 75, 469-492. ECCLES, J. C. (1964) The Physiology of Synapses. Springer: Berlin. ECCLES, J. C., FATT, P. and KOrETSU, K. (1954) Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol., Lond. 126, 524-562. ECCLES, J. C., ITO, M. and SZENTAGOTHA1, J. (1967) The Cerebellum as a Neuronal Machine. Springer: Berlin. ECCLES, J. C., NICOLL, R. A., OSHIMA, T. and RUBIA, F. J. (1977) The anionic permeability of the inhibitory postsynaptic membrane of hippocampal pyramidal cells. Proc. R. Soc. Lond. B. 198, 345-361. EDWARDS, F. R., HARRISON, P. J., JACK, J. J. B. and KULLMANN, D. M. (1989) Reduction by baclofen of monosynaptic EPSPs in lumbosacral motoneurones of the anaesthetized cat. J. Physiol., Lond. 416, 539-556. EGAN, T. M., HENDERSON,G., NORTH, R. A. and WILLIAMS, J. T. (1983) Noradrenaline-mediated synaptic inhibition in rat locus coeruleus neurones. J. Physiol., Lond. 345, 477-483. EICH1NGER, A. and SIEGHART,W. (1986) Postnatal development of proteins associated with different benzodiazepine receptors. J. Neurochem. 46, 173-180. EMSON, P. C. and LINDVALL, O. (1979) Distribution of

GABA RECEPTORMECHANISMS putative neurotransmitters in the neocortex. Neuroscience 4, 1-30. ENNA, S. J. and MAGGI, A. (1979) Biochemical pharmacology of GABAergic agonists. Life Sci. 24, 1727-1738. ENNA, S. J. and SNYDER, S. H. (1975) Properties of ~,aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res. 100, 81-97. ENNiS, M. and ASTON-JoNES,G. (1989) GABA-mediated inhibition of locus coeruleus from the dorsomedial rostral medulla. J. Neurosci. 9, 2973-2981. FALCH, E., HEDEGAARD,A., NIELSEN,L., JENSEN, B. R., HJEDS, H. and KROGSGAARD-LARSEN,P. (1986) Comparative stereostructure-activity studies on GABA A and GABA a receptor sites and GABA uptake using rat brain membrane preparations. J. Neurochem. 47, 898-903. FELIX, D. and MCLENNAN, H. (1971) The effect of bicuculline on the inhibition of mitral cells of the olfactory bulb. Brain Res. 25, 661-664. FINLEY, J. C. W., MAOERORUT,J. L., ROGER, L. J. and PETRUSZ,P. (1981) The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system. Neuroscience 6, 2173-2192. FITZPATRICK, D., PENNY, (3. R. and SCHMECHEL,D. E. (1984) (31utamic acid decarboxylase-immunoreactive neurons and terminals in the lateral geniculate nucleus of the cat. J. Neurosci. 4, 1809-1829. FONNUM, F., STORM-MATHISEN,J. and WALBERG,F. (1970) Glutamate decarboxylase in inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat. Brain Res. 20, 259-275. FONNUM, F., (3ROFOV.~,,I., RINVICK, E., STORM-MATH1SEN, J. and WALBERG,F. (1974) Origin and distribution of glutamate decarboxylase in substantia nigra of the cat. Brain Res. 71, 77-92. FONNUM, F., WALAAS, I. and IVERSEN,E. (1977) Localization of (3ABAergic cholinergic and aminergic structures in the mesolimbic system. J. Neurochem. 29, 221-230. FONNUM,F., GOTTESEELD,Z. and GROFOV~,,I. (1978) Distribution of glutamate decarboxylase, choline acetyltransferas¢ and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopallidal, striatoentopeduncular and striatonigral (3ABAergic fibres. Brain Res. 143, 125-138. Fox, S., KRN~EVI~, K., MORRIS, M. E., PULL, E. and WERMAN,R. 0978) Action of baclofen on mammalian synaptic transmission. Neuroscience 3, 495-515. FRANK, K. (1959) Basic mechanism of synaptic transmission in the central nervous system. LR.E. Trans. reed. Electron. 6, 85-88. FRANK, K. and FUORTES, M. (3. (1957) Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fedn Proc. 16, 36-40. FREUND, T. F. and ANTAL, M. (1988) GABA-containing neurons in the septum control inhibitory interneurones in the hippocampus. Nature 336, 170-173. FREUND, T. F., MARTIN, K. A. C., SMITH, A. D. and SOMOGY1, P. (1983) Glutamate decarboxylase-immunoreactive terminals of Golgi-impregnated axoaxonic cells and of presumed basket cells in synaptic contact with pyramidal neurones of the cat's visual cortex. J. comp. Neurol. 221,263-278. FRICKE, R. A. and PRINCE,D. A. (1984) Electrophysiology of dentate gyrus granule cells. J. Neurophysiol. 51, 195-209. FRIGYESl,T. L. and SZABO,J. (1975) Caudate-evoked synaptic activity in nigral neurons. Expl Neurol. 49, 123-139. FYFFE, R. E. W. and LIGHT, A. R. (1984) The ultrastructure of group Ia afferent fibre synapses in the lumbrosacral spinal cord of the cat. Brain Res. 300, 201-209. G~HWlLER, B. H. and BROWN, D. A. (1985) GABAB-receptor-activated K + current in voltage-clamped CA 3 pyramidal cells in hippocampal cultures. Proc. natn. Acad. Sci. U.S.A. 82, 1558-1562.

83

G~-xwmv~, B. H., MAuP.£a, R. and WO'mmCH, H. J. (1984) Pitrazepin, a novel GABAA antagonist. Neurosci. Lett. 45, 311-316. GALL, C. M., HENDRY,S. H., SEROOGY,K. B., JONES,E. G. and HAYCOCK,J. W. (1987) Evidence for the co-existence of GABA and dopamine in neurons of the rat olfactory bulb. J. comp. Neurol. 266, 307-318. GALVAN, M., FRANZ, P. and CONSTANTLA. (1985) Spontaneous inhibitory postsynaptic potentials in guinea-pig neocortex and olfactory cortex neurones. Neurosci. Lett. 57, 131-135. GARDNER,C. R. (1986) Blockade of a putative (3ABA-mediated neurotransmission in the cerebellum by benzodiazepine receptor inverse agonists. Comp. 8iochem. Physiol. 85C, 225-232. GARDNER,C. R., KLEIN, J. and GROW, J. 0981) Endogenous GABA determines the characteristics of [3H](3ABAbinding. Eur. J. Pharmac. 75, 83-92. GEE, K. W. (1987) Phenylqulnolines PK 8165 and PK 9084 allosterically modulate [35S]-t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5 4864 binding site. J. Pharmac. exp. Ther. 240, 747-753. GEE, K. W., YAMAMURA,S. H., ROESKE,W. R. and YAMAMURA, H. I. (1983) Benzodiazepine receptor heterogeneity: possible molecular basis and functional significance. Fedn Proc. 43, 2767-2772. GEE, K. W., BRINTON, R. E. and McEWEN, B. S. (1988) Regional distribution of a Ro5 4864 binding site that is functionally coupled to the ,/-aminobutyric acid/benzodiazepine receptor complex in rat brain. J. Pharmac. exp. Ther. 244, 379-383. GERMROTH, P., SCriWERDTFEGER,W. K. and BUHL, E. H. (1989) (3ABAergic neurons in the entorhinal cortex project to the hippocampus. Brain Res. 494, 187-192. (3ETCHELL,T. V. and SHEPHERD,G. M. (1975) Synaptic actions on mitral and tufted cells elicited by olfactory nerve volleys in the rabbit. J. Physiol., Lond. 251, 497-522. GRACE, A. A. and BUNNEY,B. S. (1985) Opposing effects of striatonigral feedback pathways on midbrain dopamine cell activity. Brain Res. 333, 271-284. GRAY, R. and JOHNSTON,D. (1985) Rectification of single (3ABA-gated chloride channels in adult hippocampal neurons. J. Neurophysiol. 54, 134-142. (3tnDOTTI, A., TOFFANO, (3. and COSTA, E. (1978) An endogenous protein modulates the affinity of (3ABA and benzodiazepine receptors in rat brain. Nature 275, 553-555. GYENES, M., FARRANT,M. and FARB, D. H. (1988) "Rundown" of (3ABAA receptor: a possible role for phosphorylation. Molec. Pharmac. 34, 719-723. HABERLY, L. B., HANSEN,D. J., FEIG, S. L. and PRESTO,S. (1987) Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake. J. comp. Neurol. 266, 269-290. HABLITZ, J. J. and THALMANN,R. H. (1987) Conductance change underlying a late synaptic hyperpolarization in hippocampal CA3 neurons. J. Neurophysiol. 58, 160-179. HALASZ,N. and SHEPHERD,(3. M. (1983) Neurocbemistry of the vertebrate olfactory bulb. Neuroscience 10, 579-619. HAL~SZ, N., IAUNGDAHL,A. and H6KFELT,T. (1979) Transmitter histochemistry of the rat olfactory bulb. III. Autoradiographic localization of [3H](3ABA. Brain Res. 167, 221-240. HARRIS, R. A. and ALLAN, A. M. (1985) Functional coupling of ~,-aminobutyric acid receptors to chloride channels in brain membranes. Science 228, 1108-1110. HARRISON, N. L. (1990) On the presynaptic action of baclofen at inhibitory synapses between cultured rat hippocampal neurones. J. Physiol., Lond. 422, 433-446. HARRISON, N. L., MAJEWSKA,M. D., HARRINGTON,J. W. and BARKER,J. L. (1987) Structure-activity relationships

84

L. SIVILOTT1and A. NISTRI

for steroid interaction with the ~-aminobutyric acid A receptor complex. J. Pharmac. exp. Ther. 242, 346-353. HASUO, H. and GALLAGHER, J. P. (1988) Comparison of antagonism by phaclofen of baclofen-induced hyperpolarizations and synaptically-mediated late hyperpolarizing potentials recorded intracellularly from rat dorsolateral septal neurons. Neurosci. Lett. 86, 77-81. HATTORI, T., MCGEER, P. L., F/alGER, H. C. and MCGEER, E. (1973) On the source of GABA-containing terminals in substantia nigra. Electron microscopic autoradiographic and biochemical studies. Brain Res. 54, 103 l l4. HAVOUNDJIAN, H., PAUL, S. M. and SKOLNICK, P. (1986) The permeability of 7-aminobutyric acid-gated chloride channels is described by the binding of a "cage" convulsant, t-butylbicyclophosphoro [35S]thionate. Proc. natn. Acad. Sci. U.S.A. 83, 9241-9244. HENDRICKSON, A. E., HUNT, S. P. and Wu, J.-Y. (1981) Immunocytochemical localization of glutamic acid decarboxylase in monkey striate cortex. Nature 292, 605-607. HENDRICKSON, A. E., OGREN, M. P., VAUGHN, J. E., BARBUR, R. P. and WE, J.-Y. (1983) Light and electron microscopic immunocytochemical localization of glutamic acid decarboxylase in monkey geniculate complex: evidence for GABAergic neurons and synapses. J. Neurosci. 3, 1245-1262. HENDRY, S. H. C., JONES, F. G., DE FELIPE, J., SCHMECHEL, D., BRANDON, C. and EMSON,P. C. (1984) Neuropeptidecontaining neurons of the cerebral cortex are also GABAergic. Proc. natn. Acad. Sci. U.S.A. 81, 6526~530. HENRY, T. A. (1949) The Plant Alkaloids. Churchill Ltd: London. HERRLING, P. L. (1984) Evidence for GABA as the transmitter for early cortically-evoked inhibition of cat caudate neurons. Expl Brain Res. 55, 528-534. HEYER, E. J., NOWAK, L. M. and MACDONALD, R. L. (1981) Bicuculline: A convulsant with synaptic and non-synaptic actions. Neurology 31, 1381 1390. HILL, D. R. and BOWERY, N. G. (1981) 3H-baclofen and 3H-GABA bind to bicuculline-insensitive GABA s sites in rat brain. Nature 290, 149-152. HIRSCH, J. C. and BURNOD, Y. (1987) A synaptically evoked late hyperpolarization in the rat dorsolateral geniculate neurones in vitro. Neuroscience 23, 457-468. HOLZ, G. G., RANE, S. G. and DUNLAP, K. (1986) GTPbinding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature 319, 670-672. HOTSON, J. R. and PRINCE, D. A. (1980) A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurones. J. Neurophysiol. 43, 409-419. HOUSER, C. R., VAUGHN, J. E., BARBER,R. P. and ROBERTS, E. (1980) GABA neurons are the major cell type of nucleus reticularis thalami. Brain Res. 2110, 341-354. HoWE, J. R., SUTOR, B. and Z1EGLGXNSBERGER,W. (1987a) Characteristics of long-duration inhibitory postsynaptic potentials in rat neocortical neurons in vitro. Cell molec. Neurobiol. 7, 1 18. HOWE, J. R., SUTOR, B. and ZIEGLG.~NSBERGER,W. (1987b) Baclofen reduces post-synaptic potentials of rat cortical neurones by an action other than its hyperpolarizing action. J. Physiol., Lond. 384, 539-569. HUGUENARD, J. R. and ALGER, B. E. (1986) Whole-cell voltage-clamp study of the fading of GABA-activated currents in acutely dissociated hippocampal neurons. J. Neurophysiol. 56, 1-18. HUIDOBRO-ToRo, J. P., BLECK, V., ALLAN, A. M. and HARRIS, R. A. (1987) Neurochemical actions of anesthetic drugs on the y-aminobutyric acid receptor-chloride channel complex. J. Pharmac. exp. Ther. 242, 963-969. HULL, C. n., BERNARDI, G. and BUCHWALD, N. A. (1970) Intracellular responses of caudate neurons to brain stem stimulation. Brain Res. 22, 163-179.

HUNT, S. P. (1983) Cytochemistry of the spinal cord. In: Chemical Neuroanatomy, pp. 53-84. Ed. P. C. EMSON. Raven Press: New York. IM, W. B., TAI, M. M., BLAKEMAN,D. P. and DAVIS, J. P. (1989) Immobilized GABA A receptors and their ligand binding characteristics. Biochem. biophys. ICes. Commun. 163, 611-617. INOUE, M., MATSUO, T. and OGATA, N. (1985) Characterization of pre- and postynaptic actions of (-)-baclofen in the guinea-pig hippocampus in vitro. Hr. J. Pharmac. 84, 843-851. INOUE, M., OOMURA, Y,, YAKUSHIJI, T. and AKAIKE, N. (1986) Intracellular calcium ions decrease the affinity of the GABA receptor. Nature 324, 156-158. ITO, M. and YOSmDA, M. (1966) The origin ofcerebellar-induced inhibition of Deiters neurones. I. Monosynaptic initiation of the inhibitory postsynaptic potentials. Expl Brain Res. 2, 330-349. JAHR, C. E. and NICOLL, R. A. (1982) An intracellular analysis of dendrodendritic inhibition in the turtle in vitro olfactory bulb. J. Physiol., Lond. 326, 213-234. JOHNSTON, D. and BROWN, T. H. (1983) Interpretation of voltage-clamp measurements in hippocampal neurons. J. Neurophysiol. 50, 464-486. JOHNSTON,G. A. R., HEART,P. M., CURTIS,D. R., GAME, C. J. A., MCCULLOCH, R. M. and MACLACHLAN, R. M. (1972) Bicuculline methochloride as a GABA antagonist. Nature (New Biol.) 240, 219-220. JONES, E. G. (1985) The Thalamus. Plenum Press: New York. JONES, E. G. and HENDRY, S. H. C. (1986) Co-localization of GABA and neuropeptides in neocortical neurons. Trends Neurosci. 9, 71-76. KAILA, K. and VolPlO, J. (1987) Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance. Nature 330, 163-165. KAILA, K., PASTERNACK, M., SAARIKOSKI,J. and VOlPIO, J. (1989) Influence of GABA-gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibres. J. Physiol., Lond. 416, 161-181. KANDEL, E. R., SPENCER, W. A. and BRINLEY, F. J. (1961) Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization. J. Neurophysiol. 24, 225-242. KANEDA, M., WAKAMORI, M. and AKAIKE, N. (1989) GABA-induced chloride current in rat isolated Purkinje cells. Am. J. Physiol. 256, Cl153-1159. KARLSSON, (~. and OLPE, H. R. (1989) Late inhibitory postsynaptic potentials in rat prefrontal cortex may be mediated by GABA s receptors. Experientia 45, 157-I 58. KARLSSON, G., POZZA, M. and OLPE, H. R. (1988) Phaclofen: a GABA a blocker reduces long-duration inhibition in the neocortex. Fur. J. Pharmac. 148, 485-486. KAROBATH, M., DREXLER, G. and SUPAVILAI,P. (1981) Modulation by picrotoxin and IPTBO of 3H-flunitrazepam binding to the GABA/benzodiazepine receptor complex of rat cerebellum. Life Sci. 28, 307-313. KATZ, B. and MILEDI, R. (1972)The statistical nature of the acetylcholine potential and its molecular components. J. Physiol., Lond. 224, 665-699. KAWAGUCHI, S. and ONO, T. (1973) Bicuculline and picrotoxin-sensitive inhibition in interpositus neurones of cat. Brain Res. 58, 260-265. KELLY, J. S., KRNJEVI(~, K., MORRIS, M. E. and YIM, G. K. W. (1969) Anionic permeability of cortical neurones. Expl Brain Res. 7, l 1-31. KEMP, J. A., MARSHALL,G. R. and WOODRUFF,G. N. (1986) Quantitative evaluation of the potencies of GABA-receptor agonists and antagonists using the rat hippocampal slice preparation. Br. J. Pharmac. 87, 677-684. KERR, D. I. B., ONG, J., PRAGER, R. H., GYNTHER, B. D. and CURTIS, D. R. (1987) Phaclofen: a peripheral and central baclofen antagonist. Brain Res. 405, 150-154.

GABA RECEPTORMECHANISMS KERR, D. I. B., ONG, J., JOHNSTON,G. A. R., ABBENANTE, J. and PRAGER, R. H. (1988) 2-Hydroxysaclofen: an improved antagonist at central and peripheral GABAB receptors. Neurosci. Lett. 92, 92-96. KIRKNESS,E. F., BOVENKERK,C. F., UEDA,T. and TURNER, A. J. (1989) Phosphorylation of ~,-aminobutyrate (GABA)/benzodiazepine receptors by cyclic AMP-dependent protein kinase. Biochem. J. 259, 613-616. KITA, T., KITA, H. and KITAI,S. T. (1985) Local stimulation induced GABAergic response in rat striatal slice preparations: intracellular recordings on QX-314 injected neurons. Brain Res. 360, 304-310. KZTAI,S. T. (1981) Electrophysiology of the corpus striatum and brainstem integrating systems. In: Handbook of Physiology, Section I: The Nervous System Vol. II, Motor Control, Part 2, pp. 997-1016. Eds. J. M. BROOIOtARTand V. B. MOUNTCASTLE.American Physiological Society: Bethesda. KITA1, S. T. and KITA, H. (1984) Electrophysiological study of the neostriatum in brain slice preparation. In: Brain Slices, pp. 285-296. Ed. R. DINGLED1NE.Plenum Press: New York and London. KLEPNER, C. A., LIPPA, A. S., BENSON,D. I., SAND, M. C. and BEER, B. (1979) Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors. Pharmac. Biochem. Behav. 11, 457-462. KNOWLES, W. D. and SCHWARTZKROIN,P. A. (1981) Local circuit interactions in hippocampal brain slices. J. Neurosci. I, 318-322. KNOWLES, W. D., SCHNEIDERMAN,J. H., WHEAL, H. V., STAFSTROM, C. E. and SCHWARTZKROIN,P. A. (1984) Hyperpolarizing potentials in guinea pig hippocampal CA3 neurons. Cell molec. Neurobiol. 4, 207-230. K6HLER, C., CHAN-PALAY,V. and Wu, J.-Y 0984) Septal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat. Brain Anat. Embryol. 169, 41-44. KOSAKA, T., HATAGUCHI,Y., HAMA, K., NAGATSU, I. and Wu, J.-Y. (1985) Co-existence of immunoreactivities for glutamate decarboxylase and tyrosine hydroxylase in some neurons in the perigiomerular region of the rat main olfactory bulb: possible co-existence of gamma-aminobutyric acid (GABA) and dopamine. Brain Res. 343, 166-171. KOSAKA, T., KOSAKA,K., HEIZMANN,C. W., NAGATSU,I., WU, J.-Y., YANAmARA, N. and HAMA, K. (1987) An aspect of the organization of the GABAergic system in the rat main olfactory bulb: laminar distribution of immunohistochemically defined subpopulations of GABAergic neurons. Brain Res. 411, 373-378. KRNJEVI6, K. (1981) Desensitization of GABA receptors. Adv. Biochem. Psychopharmac. 26, 111-120. KRNJEVI~, K. (1982) GABA and other transmitters in the cerebellum. In: The Cerebellum. New Vistas, pp. 532-551. Eds. S. L. PALAYand V. CHAN-PALAY.Springer Verlag: Berlin/Heidelberg. KRNJEVI(:, K. and PHILLIS, J. W. (1963) Iontophoretic studies of neurones in the mammalian cerebral cortex. J. Physiol., Lond. 165, 274-304. KRNJEVI(~, K. and SCHWARTZ, S. (1967) The action of ),-aminobutyric acid on cortical neurones. Expl Brain Res. 3, 320-336. KRNJEVI(~, K., RANDI(~,M. and STRAUGHAN,D. W. (1964) Cortical inhibition. Nature 201, 1294-1296. KRNJEV]~, K., RANm~, M. and STRAUGHAN,D. W. (1966a) An inhibitory process in the cerebral cortex. J. Physiol., Lond. 184, 16-48. KRNJEV]~, K., RANDI~, M. and STRAUGHAN,D. W. (1966b) Nature of a cortical inhibitory process. J. Physiol., Lond. 184, 49-77, KROGSGAARD-LARSEN, P., FALCH, E. and JACOBSEN, P. (1984) GABA agonists: structural requirements for interaction with the GABA-benzodiazepine receptor complex.

85

In: Actions and Interactions of GABA and Benzodiazepines, pp. 109-132. Ed. N. G. BOWERY.Raven Press: New York. KUmYAMA, K., HABER, B., SISKEN, B. and ROBERTS, E. (1966) The ),-aminobutyric acid system in rabbit cerebellum. Proe. narD. Acad. Sci. U.S.A. 55, 846-852. LACEY,M. G., MERCUR1,N. B. and NORm, R. A. (1988) On the potassium conductance increase activated by GABA B and dopamine D 2 receptors in rat substantia nigra neurones. J. Physiol., Lond. 401, 437--453. LAMBERT,N. A., HARRISON,N. L., KERR, D. I. B., ONG, J., PRAGER, R. H. and TEYLER,T. J. (1989) Blockade of the late IPSP in rat CA1 hippocampal neurons by 2-hydroxysaclofen. Neurosci. Lett. 107, 125-128. LARSON, M. D. (1969) An analysis of the action of strychnine on the recurrent IPSP and amino acid induced inhibitions in the cat spinal cord. Brain Res. 15, 185-200. LARSON, M. D. and MAJOR, M. A. (1970) The effect of hexobarbital on the duration of the recurrent IPSP in cat motoneurons. Brain Res. 21, 309-311. LAWRENCE, L. J. and CASIDA,J. E. (1983) Stereospecific action of pyrethroid insecticides on the 7-aminobutyric acid receptor-ionophore complex. Science 221, 13991341. LEBEDA, F. J., HABLITZ, J. J. and JOHNSTON, D. (1982) Antagonism of GABA-mediated responses by dtubocurarine in hippocampal neurons. J. Neurophysiol. 40, 622-632. LEEB-LUNDBERG, F., SNOWMAN, A. and OLSEN, R. W. (1980) Barbiturate receptor sites are coupled to benzodiazepine receptors. Proc. hath. Acad Sci. U.S.A. 77, 7468-7472. LEFUR, G., PERRIER, M. L., VAUCHER,N., IMBAULT, F., FLAMIER, A., BENAVIDES,J., UZAN, A., RENAULT, C., DUBROECQ, M. C. and GU~R~MY, C. (1983) Peripheral benzodiazepine binding sites: effect of PK ! 1195, 1-(2chloropbenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide. I. In vitro studies. Life Sci. 32, 1839-1847. LEHOULLIER,P. F. and TICKU, M. K. (1989) The pharmacological properties of GABA receptor-coupled chloride channels using 36Cl-influx in cultured spinal cord neurons. Brain Res. 487, 205-214. LEWTAN, E., SCHOFmLD,P. R., BURT, D. R., RrmE, L. M., WISDEN,W., K6HLVaLM., FUJITA,N., RODRIGUEZ,H. F., STEPHENSON,A., DARLISON,M. G., BARNARD,E. A. and SEEBURG,P. H. (1988) Structural and functional basis for GABAA receptor heterogeneity. Nature 335, 76-79. LIGHTHALL,J. W. and KITAI, S. T. (1983) A short duration GABAergic inhibition in identified neostriatal medium spiny neurons: in vitro slice study. Brain Res. Bull. 11, 103-110. LIGHTHALL, J. W., PARK, M. R. and KITAI, S. T. (1981) Inhibition in slices of rat neostriatum. Brain Res. 212, 182-187. LLANO, I., MARTY, A., JOHNSON, J. W., ASCHER, P. and GXHWmER, B. H. (1988) Patch-clamp recording in "organotypic" slice cultures. Proc. Dam. Acad. Sci. U.S.A. 85, 3221-3225. LLINAS, R. (1981) Electrophysiology of the cerebellar networks. In: Handbook o f Physiology, Section 1: The Nervous System. Vol. II, Motor Control, Part 2, pp. 831-876. Eds. J. M. BROOKHARTand V. B. MOUNTCASTLE,American Physiological Society: Bethesda. LLOYD, K. G. and BEAUMONT,K. (1980) Possible role of phospholipids in GABA receptor function in human and rat brain. Brain Res. Bull. 5(suppl. 2), 285-290. MACDONALD, J. F., MODY, I. and SALTER, M. W. (1989) Regulation of N-methyl-D-aspartate receptors revealed by intracellular dialysis of murine neurones in culture. J. Physiol., Lond. 414, 17-34. MACDONALD,R. L. and BARKER,J. L. (1977) Pentylenetetrazol and penicillin are selective antagonists of GABA-

86


mediated post-synaptic inhibition in cultured mammalian neurones. Nature 267, 720-721. MACDONALD, R. L., ROGERS, C. J. and TWYMAN, R. E. (1989a) Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J. Physiol., Lond. 410, 479-499. MACDONALD, g. L., ROGERS, C. J. and TWYMAN, R. E. (1989b) Barbiturate regulation of kinetic properties of the GABA A receptor channel of mouse spinal neurones in culture. J. Physiol., Lond. 417, 483-500. MAcVICAR, B. A. and DUDEK, F. E. (1980) Local synaptic circuits in rat hippocampus: interactions between pyramidal cells. Brain Res. 184, 220-223. MAcVICAR, B. A., TSE, F. W. Y., CRICHTON, S. A. and KETTENMANN, H. (1989) GABA-activated C1- channels in astrocytes of hippocampal slices. J. Neurosei. 9, 3577-3583. MADARASZ, M., SOMOGYI, G., SOMOGY1,J. and HAMORI, J. (1985) Numerical estimation of 7-aminobutyric acid (GABA)-containing neurons in three thalamic nuclei of the cat: direct GABA immunocytoehemistry. Neurosci. Lett. 61, 73 78. MAJEWSKA, M. D., HARRISON, N. L., SCHWARTZ, R. D., BARKER, J. L. and PAUL, S. M. (1986) Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232, 1004-1007. MARTIN, 1. L., BROWN, C. L. and DOBLE, A. (1983) Multiple benzodiazepine receptors: structures in the brain or structures in the mind? A critical review. Life Sci. 32, 1925-1933. MARTIN, K. A. C. (1988) The lateral geniculate nucleus strikes back. Trends Neurosci. 11, 192-194. MATHERS, D. A. (1985) Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons. Can. J. Physiol. Pharmac. 63, 1228-1233. MATHERS, D. A. (1987) The GABA A receptor: new insights from single-channel recording. Synapse 1, 96-101, MATHERS, D. A. and BARKER,J. L. (1980) (--) Pentobarbital opens channels of long duration on cultured mouse spinal neurons. Science 209, 507-509. MATSUMOTO, R. R. (1989) GABA receptors: are cellular differences related to function? Brain Res. Rev. 14, 203-225. MAZDA, G. Y., NISTRI, A. and SIVILOTTI, L. (1990) The effect of GABA on the frog optic rectum is sensitive to ammonium and to penicillin. Eur. J. Pharmac. 179, I11 118. McBRIDE, W. J. and FREDERICKSON, R. C. A. (1980) Taurine as a possible inhibitory transmitter in the cerebellum. Fedn Proc. 39, 2701-2705. McBURNEY, R. N. and BARKER,J. L. (1978) GABA-induced conductance fluctuations in cultured spinal neurons. Nature 274, 59(~597. MCCARREN, M. and ALGER, B. E. (1985) Use-dependent depression of IPSPs in rat hippocampal pyramidal cells in vitro. J. Neurophysiol. 53, 557-571. McCORMICK, D. A., CONNORS, B. W., LIGHTHALL,J. W. and PRINCE, D. A. (1985) Comparative physiology of pyramidal and sparsely spiny neurons of the neocortex. J. NeurophysioL 54, 782-806. MCGEER, P. L., MCGEER, E. G., WADA, J. A. and JUNG, E. (1971) Effect of globus pallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase. Brain Res. 32, 425 431. McLAUGHLIN, B. J., WOOD, J. G., SAITO, K., BARBER, R., VAUGHN, J. E., ROBERTS,E. and WU, J.-Y. (1974) The fine structural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum. Brain Res. 76, 377-391. MCLAUGHLIN, B. J., BARBER, R., SAITO, K., ROBERTS, E. and WE, J.-Y. (1975) Immunocytochemical localization of glutamate decarboxylase in rat spinal cord. J. eomp. Neurol. 164, 305 332.

McLENNAN, H. (1971) The pharmacology of inhibition of mitral cells in the olfactory bulb. Brain Res. 29, 177-184. McLENNAr~, H. and MILLER, J. J. (1974a) The hippocampal control of neuronal discharges in the septum of the rat. J. Physiol., Lond. 237, 607-624. MCLENNAN, H. and MILLER, J. J. (1974b) ~-Aminobutyric acid and inhibition in the septal nuclei of the rat. J. Physiol., Lond. 237, 625-633. MEHTA, A. K. and T1CKU, M. K. (1989) Benzodiazepine and beta-carboline interactions with GABA A receptor-gated chloride channels in mammalian cultured spinal cord neurons. J. Pharmac. exp. Ther. 249, 418-423. MILES, R. and WONG, R. K. S. (1984) Unitary inhibitory synaptic potentials in the guinea-pig hippocampus in vitro. J. Physiol., Lond. 356, 97-113. MISGELD, U. and FROTSCHER, M. (1986) GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus. Neuroscience 19, 193-206. MISGELD, O., OKADA, Y. and HASSLER, R. (1979) Locally evoked potentials in slices of rat neostriatum: a tool for the investigation of intrinsic excitatory processes. Expl Brain Res. 34, 575-590. MISGELD, O., WAGNER, A. and OHNO, T. (1982) Depolarizing IPSPs and depolarization by GABA of rat neostriatum cells in vitro. Expl Brain Res. 45, 108-114. MISGELD, U., KLEE, M. R. and ZEISE, M. L. (1984) Differences in baclofen-sensitivity between CA3 neurons and granule cells of the guinea pig hippocampus in vitro. Neurosci. Lett. 47, 307 311. MISGELD, U., DEISZ, R. A., DODT, H. V. and Lux, H. D. (1986) The role of chloride transport in postsynaptic inhibition of hippocampal neurons. Science 232, 1413 1415. MISTRY, D. K. and HABLITZ, J. J. (1989) Single-channel currents activated by GABA, muscimol and isoguvacine in outside-out membrane patches from cultured chick cerebral neurons. Soc. Neurosci. Abstr. 15, 970. MITCHELL, P. R. and MARTIN, I. L. (1978) Is GABA release modulated by presynaptic receptors? Nature 274, 904-905. MOFFETT, J. R., NAMBOODIRI, A. and NEALE, J. H. (1989) Two forms of the GABAA receptor distinguished by anion-exchange chromatography. FEBS Lett. 247, 81-85. M6HLER, H. and OKADA, T. (1977) Benzodiazepine receptor: demonstration in the central nervous system. Science 198, 849-851. M6HLER, H. and RICHARDS, J. G. (1981) Agonist and antagonist benzodiazepine receptor interaction in vitro. Nature 294, 763-765. MGHLER, H., MALHERBE, P. and DRAGUHN, A. (1989) GABAA-receptors expressed from rat ~- and/~-subunits in Xenopus oocytes are modulated by benzodiazepine receptor ligands. Soc. Neurosci. Abstr. 15, 997. MONTERO, V. (1986) Localization of ~-aminobutyric acid (GABA) in type 3 cells and demonstration of their source to F2 terminals in the cat lateral geniculate nucleus: A Golgi-elect ronmicr oscopic GABA-immunocytochemical study. J. comp. Neurol. 254, 228-245. MONTERO, V. and SINGER, W. (1984) Ultrastructure and synaptic relations of neural elements containing glutamic acid decarboxylase (GAD) in the perigeniculate nucleus of the cat. Expl Brain Res. 56, 115-125. MORI, K. (1987) Membrane and synaptic properties of identified neurons in the olfactory bulb. Prog. Neurobiol. 29, 275-320. MORI, K. and TAKAG1,S. F. (1978a) An intracellular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit olfactory bulb. J. Physiol., Lond. 279, 569-588. MORI, K. and TAKAGI, S. F. (1978b) Activation and inhibition of olfactory bulb neurones by anterior commissure volleys in the rabbit. J. Physiol., Lond. 729, 589-604. MORI, K., NOWYCKY, M. C. and SHEPHERD, G. M. (1981a) Analysis of synaptic potentials in mitral cells in the

GABA RECEPTORMECHANISMS isolated turtle olfactory bulb. J. Physiol., Lond. 314, 295-309. Morn, K., NowvcKv, M. C. and SHEPHERD,G. M. (1981b) Analysis of a long-duration inhibitory potential in mitral cells in the isolated turtle olfactory bulb. J. Physiol., Lond. 314, 311-320. Morn, K., Klsal, K. and OJBaA, H. (1983) Distribution of dendrites of mitral, displaced mitral, tufted and granule cells in the rabbit olfactory bulb. J. comp. Neurol. 219, 339-355. MUGNAINI, E., WOUTERLOOD, F. G., DArtL, A. L., and OERTEL, W. H. (1984a) Immunocytochemical identification of GABAergic neurons in the main olfactory bulb of the rat. Archs ital. Biol. 122, 83-113. MUGNAINI, E., OERTEL, W. H. and WOUTERLOOD,F. G. (1984b) Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulb. Neurosci. Lett. 47, 221-226. Mf~LLER, W. and MISGELD, U. (1989) Carbachol reduces IK.hc,or~,, but not IK.CABA in guinea-pig bippocampal slices. Neurosci. Lett. 102, 229-234. NAGAI, T., McGEER, P. L. and MCGEER, E. G. (1983) Distribution of GABA-T intensive neurons in the rat forebrain and midbrain. J. comp. Neurol. 218, 220-238. NANOPOULOS,D., BELIN, M. F., VICENDON,G. and PUJOL, J. F. (1982). Immunocytochemical evidence for the existence of GABA-ergic neurons in the nucleus raphe dorsalis. Possible existence of neurons containing serotonin and GABA. Brain Res. 232, 375-389. NELSON,P. G., RANSOM,B. R., HENKART,M. and BULLOCK, P. N. (1977) Mouse spinal cord in cell culture, IV. Modulation of inhibitory synaptic function. J. Neurophysiol. 40, 1178-1187. NEWBERRY,N. R. and NICOLL, R. A. (1984a) Direct hyperpolarizing action of baclofen on hippocampal pyramidal cells. Nature 308, 450-452. NEWBERRY,N. R. and NICOLL, R. A. (1984b) A bicucullineresistant inhibitory post-synaptic potential in rat hippocampal pyramidal cells in vitro. J. Physiol., Lond. 348, 239-254. NEWBERRY,N. R. and NICOLL, R. A. (1985) Comparison of the action of baclofen with ~/-aminobutyric acid on rat hippocampal pyramidal cells in vitro. J. Physiol., Lond. 360, 161-185. NICOLL, R. A. (1971) Pharmacological evidence for GABA as transmitter in granule cell inhibition in the olfactory bulb. Brain Res. 35, 137-149. NICOLL, R. A. (1975) Presynaptic action of barbiturates in the frog spinal cord. Proc. ham. Acad. Sci. U.S.A. 72, 1460-1463. NICOLL, R. A., ECCLES, J. C., OSmMA, T. and RUBIA, F. (1975) Prolongation of hippocampal inhibitory postsynaptic potentials by barbiturates. Nature 258, 625-627. NIEUWENHUYS, R. (1985) Chemoarchitecture of the Brain. Springer-Vcrlag: Berlin, Heidelberg. NISTRI, A. (1975) Further investigations into the effects of baclofen (Lioresal) on the isolated spinal cord. Experientia 31, 1066-1067. NISTRL A. (1983) Spinal cord pharmacology of GABA and chemically related amino acids. In: Handbook of the Spinal Cord, Vol. 1 Pharmacology, pp. 45-104. Ed. R. A. DAVIDOFF. Dekker: New York. NISTRI, A. and CONSTANTI, A. (1979) Pharmacological characterization of different types of GABA and glutamate receptors in vertebrates and invertebrates. Prog. Neurobiol. 13, 117-235. NISTRI, A. and SIVILOTTI, L. (1985) An unusual effect of ),-aminobutyric acid on synaptic transmission on frog tectal neurones in vitro. Br. J. Pharmac. 85, 917-922. NISTRI, A. and SIVILOTTI, L. (1988) Effects of GABA and related drugs on the compound action potential of the frog optic nerve in vitro. J. Physiol., Lond. 396, 146P.

87

NORTHMORE,D. P. M., L~VINE,E. S. and SCHNEIDER,G. E. (1988) Behaviour evoked by electrical stimulation of the hamster superior collieulus. Expl Brain Res. 73, 595-605. NOWYCKY, M. C., MoP,I, K. and SrEPH~gD, G. M. (1981) GABAergic mechanisms of dendrodentritic synapses in isolated turtle olfactory bulb. J. Neurophysiol. 46, 639-648. OBATA,K., ITO, M., OCm, R. and SATO,N. (1967) Pharmacological properties of the postsynaptic inhibition by Purkinje cell axons and the action of ~-aminobutyric acid on Deiters neurones. Expl Brain Res. 4, 43-57. OBATA, K., TAKEDA,K. and SHINOZAKI,H. (1970) Further study on pharmacological properties of the cerebelluminduced inhibition of Deiters neurones. Expl Brain Res. 11, 327-342. OBATA, T. and YAMAMURA,H. I. (1986) The effect of benzodiazepines and fl-carbolines on GABA-stirnulated chloride influx by membrane vesicles from the rat cerebral cortex. Biochem. biophys. Res. Commun. 141, 1-6. OERTEL, W. H. and MUGNAIN1, E. (1984) Immunocytochemical studies of GABAergic neurons in rat basal ganglia and their relation to other neuronal systems. Neurosci. Lett. 47, 233-238. OERTEL, W. H., SCHMECHEL,D. E., MUGNAINI,E., TAPPAZ, M. L. and KOPIN, I. J. (1981) Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6, 2715-2735. OERTEL, W. H., GRAYBIEL,A. M., MUGNAINI,E., ELDE, R. P., SCHMECtlEL,D. E. and KOPIN, I. J. (1983) Coexistence of giutamic acid decarboxylase and somatostatin-like immunoreactivity in neurons of the nucleus reticularis thalami. J. Neurosci. 3, 1322-1332. OGATA, N., INOUE, M. and MATSUO,T. (1987) Contrasting properties of K + conductances induced by baclofen and y-aminobutyric acid in slices of the guinea-pig hippocampus. Synapse 1, 62-69. OGDEN, D. C. and STANF1ELD,P. R. (1987) Introduction to single channel recording. In: Microelectrode Techniques. The Plymouth Workshop Handbook, pp. 63-81. Eds. N. B. STANDEN, P. T. A. GRAY and M. J. WHITAKER. The Company of Biologists Ltd: Cambridge. OHARA,P. T., LIEBERMAN,A. R., HUNT, S. P. and Wu, J.-Y. (1983) Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat: immunohistochemical studies by light and electron microscopy. Neuroscience 8, 189-212. OLSEN, R. W. (1982) Drug interactions at the GABA receptor-ionophore complex. A. Rev. Pharmac. Toxicol. 22, 245-277. OLSEN, R. W., BAN, M. and MILLER, T. (1976) Studies on the neuropharmacological activity of bicuculline and related compounds. Brain Res. 102, 283-299. OLSEN, R. W., WONG, E. H. F., STAUBER,G. B. and KING, R. G. (1984) Biochemical pharmacology of the ~aminobutyric acid receptor/ionophore protein. Fedn Proc. 43, 2773-2778. OLSEN, R. W., YANG, J., KING, R. G,, DILBER,A., STAUBER, G. B. and RANSOM,R. W. (1986) Barbiturate and benzodiazepine modulation of GABA receptor binding and function. Life Sci. 39, 1969-1976. OSMANOVI~, S. S. and SHEFNER, S. A. (1988) Baclofen increases the potassium conductance of rat locus coeruleus neurons recorded in brain slices. Brain Res. 438, 124-136. OSMANOVI~,S. S. and SHEFNER,S. A. (1990) ~-Aminobutyric acid responses in rat locus coeruleus neurones in vitro: a current-clamp and voltage-clamp study. J. Physiol. Lond, 421, 15t-170. OTIS, L. C., MADISON,D. V. and NICOLL, R. A. (1985) Folio acid has a disinhibitory action in the rat hippocampal slice preparation. Brain Res. 346, 281-286. OTTERSEN, O, P. and STORM-MATHlSEN, J. (1984) Glutamate- and GABA-containing neurons in the mouse and

88


rat brain, as demonstrated with a new immunocytochemical technique. J. comp. Neurol. 229, 374-392. PADJEN, A. L. and HASmGUCm, T. (1983) Primary afferent depolarization in frog spinal cord is associated with an increase in membrane conductance. Can. J. Physiol. Pharmac. 61, 626-631. PALACIOS,J. M., WALMSLEY,J. K. and KUHAR, M. J. (1981) High affinity GABA receptors--autoradiographic localization. Brain Res. 222, 285-307. PAN, Z. Z. and WILLIAMS, J. T. (1989) GABA- and glutamate-mediated synaptic potentials in rat dorsal raphe neurons in vitro. J. Neurophysiol. 61, 719-726. PANULA, P., REVUELTA,A. V., CHENEY, D. L., Wu, J.-Y. and COSTA, E. (1984) An immunohistochemical study on the location of GABAergic neurons in rat septum. J. comp. Neurol. 222, 69-80. PARK, M. R., LIGHTHALL, J. W. and KITAI, S. T. (1980) Recurrent inhibition in the rat neostriatum. Brain Res. 194, 359-369. PENNEY, J. B. JR and YOUNG, A. B. (1981) GABA as the pallidothalamic neurotransmitter: Implications for basal ganglia function. Brain Res. 207, 195-199. PENNY, G. R., FITZPATRICK, D., SCHMECHEL, D. E. and DIAMOND, I. T. (1983) Glutamic acid decarboxylaseimmunoreactive neurons and horseradish peroxidaselabelled projection neurons in the ventral posterior nucleus of the cat and Galago senegalensis J. Neurosci. 3, 1868-1887. PENNY, G. R., AFSHARPOUR, S. and KITAI, S. T. (1986) The glutamate decarboxylase, leucine enkephalin-, methionine enkephalin- and substance P-immunoreactive neurons in the neostriatum of the rat and cat: evidence for partial population overlap. Neuroscience 17, 1011-1045. PERRY, T. L. and HANSEN, S. (1978) Biochemical effects in man and rat of three drugs which can increase brain GABA content. J. Neurochem. 30, 679-684. PHILLIPS,C. G. (1956) Intracellular records from Betz cells in the cat. Q. Jl exp. Physiol. 41, 58-69. PHILLIPS, C. G., POWELL, T. P. S. and SHEPHERD, G. M. (1963) Responses of mitral cells to stimulation of the lateral olfactory tract in the rabbit. 3". Physiol., Lond. 168, 65-88. PICKLES, H. G. and SIMMONDS,M. A. (1980) Antagonism by penicillin of ~,-aminobutyric acid depolarizations at presynaptic sites in rat olfactory cortex and cuneate nucleus in vitro. Neuropharmacology 19, 35-38. PIERAU, F.-K. and Z1MMERMAN, P. (1973) Action of a GABA-derivative on postsynaptic potentials and membrane properties of cat's spinal motoneurones. Brain Res. 54, 376-380. PITTALUGA, A. and RAITERI, M. (1987) Choline increases endogenous GABA release in rat hippocampus by a mechanism sensitive to hemicholinium-3. NaunynSchmiedebergs Arch. Pharmac. 336, 327-33 I. PITTALUGA, A. and RAITERI, M. (1988) Clonidine enhances the release of endogenous 7-aminobutyric acid through alpha-2 and alpha-I presynaptic adrenoceptors differentially located in rat cerebral cortex subregions. J. Pharmac. exp. Ther. 245, 682-686. PoLe, P. (1988) Electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action. Prog. Neurobiol. 31, 349-423. POLe, P. and HAEEELY, W. (1982) Benzodiazepines enhance the bicuculline-sensitive part of recurrent Renshaw inhibition in the cat spinal cord. Neurosci. Lett. 28, 193-197. POLLEN, D. A. and Lux, H. D (1966) Conductance changes during inhibitory postsynaptic potentials in normal and strychninized cortical neurons. J. Neurophysiol. 29, 369-381. POTASHNER, S. J. (1979) Baclofen: effects on amino acid release and metabolism in slices of guinea-pig cerebral cortex. J. Neurochem. 32, 103-109.

PRECHT, W. and YOSHIDA, M. (1971) Blockage of caudateevoked inhibition of neurons in the substantia nigra by picrotoxin. Brain Res. 32, 229-233. PRITCHETT, D. B., SONTHEIMER, H., GORMAN, C. M., KETTENMANN, H., SEEBURG,P. H. and SCHOFIELD, P. R. (1988) Transient expression shows ligand gating and allosteric potentiation of GABA A receptor subunits. Science 242, 1306-1309. PRITCHETT, D. B., SONTHEIMER, H., SHIVERS, B. D., YMER, S., KETTENMANN, H., SCHOFIELD, P. R. and SEEBURG, P. H. (1989a) Importance of a novel GABA A receptor subunit for benzodiazepine pharmacology. Nature 338, 582-585. PRITCHETT, D. B., LUDDENS, H. and SEEBURG,P. H. (1989b) Type I and Type II GABAA-benzodiazepine receptors produced in transfected cells. Science 245, 1389-1392. PROCTOR, W. R., MYNLIEFF, i . and DUNW1DOIE, T. V. (1986) Facilitatory action of etomidate and pentobarbital on recurrent inhibition in rat hippocampal pyramidal neurons. J. Neurosci. 6, 3161-3168. PUIA, G., SANTI, M. R., VICINI, S., PRITCHETT, D. B., SEEBURG, P. and COSTA, E. (1989) 4'-Chlorodiazepam decreases function of Cl- channels coupled to native and transiently expressed GABA A receptor subunits in a manner insensitive to flumazenil. Soc. Neurosci. Abstr. 15, 641. PURPURA, D. P. and COHEN, B. (1962) Intracellular recording from thalamic neurons during recruiting response. J. NeurophysioL 25, 621-635. RAITERI, M., BONANNO,G. and FEDELE, E. (1989a) Release of ~-[3H]aminobutyric acid (GABA) from electrically stimulated rat cortical slices and its modulation by GABA a autoreceptors. J. Pharmac. exp. Ther. 250, 648-653. RAITERI, M., PELLEGRINI,G., CANTONI, C. and BONANNO,G. (1989b) A novel type of GABA receptor in rat spinal cord? Naunyn-Schmiedebergs Arch. Pharmac. 340, 666~70. RALL, W. and SHEPHERD, G. M. (1968) Theoretical reconstruction of field potentials and dendrodendritic synaptic interactions in olfactory bulb. J. Neurophysiol. 31, 884--915. RAMANJANEYULU, R. and TICKU, M. K. (1984) Binding characteristics and interactions of depressant drugs with [35S] t-butyl-bicyclophosphorothionate, a ligand that binds to the picrotoxin site. J. Neurochem. 42, 221-229. REDMANN, G. A. and BARKER, J. L. (1984) Diazepam and voltage increase GABA activated CI- ion channel opening kinetics in cultured mouse spinal neurons. Soc. Neurosci. Abstr. 10, 642. RENAUD, L. P., KELLY, J. S. and PROVINI, L. (1974) Synaptic inhibition in pyramidal tract neurons: membrane potential and conductance changes evoked by pyramidal tract and cortical surface stimulation. J. Neurophysiol. 37, 1144-1155.

RENSrIAW, B. (1941) Influence of discharge of motoneurons upon excitation of neighbouring motoneurons. J. Neurophysiol. 4, 167-183. RIBAK, C. E. (1978) Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase. J. Neurocytol. 7, 461-478. RIBAK, C. E., VAUGHN, J. E., SAITO, K., BARBER, R. and ROBERTS, E. (1976) Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra. Brain Res. 116, 287-298. RIBAK, C. E., VAUGHN, J. E., SAITO, K., BARBER, R. and ROBERTS, E. (1977) Glutamate decarboxylase localization in neurons of the olfactory bulb. Brain Res. 126, 1-18. RIBAK, C. E., VAUGHN,J. E. and SAITO, K. (1978) Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport. Brain Res. 140, 315-332. RIBAK, C. E., VAUGHN, J. E. and ROBERTS, E. (1979) The GABA neurons and their axon terminals in rat corpus

GABA

RECEPTORMECHANISMS

striatum as demonstrated by GAD immunocytochemistry. J. comp. Neurol. 187, 261-284. RICHTER, J. A. and CURTIS, L. R. (1988) Effects of basal and phenobarbital-enhanced specific GABA binding to rat brain membranes /n vitro. Soc. Neurosci. Abstr. 15, 1153. RILEY, M. and SCHOLFIELD,C. N. (1983) Diazepam increases GABA mediated inhibition in the olfactory cortex slice. Pfliigers Arch. 397, 312-318. ROBINSON, T. N., CROSS, A. J.,GREEN, A. R., TOCZEK, J. M. and BOAR, B. R. (1989) Effectsof the putativeantagonists phaclofen and 6-aminovaleric acid on G A B A e receptor biochemistry. Br. J. Pharmae. 98, 833--840. ROGERS, C. J. and MACDONALD, R. L. (1986) Diazepam increasesthe burst frequency of GABA-activated chloride channels. Soc. Neurosci. Abstr. 12, 669. ROVIRA, C., GHO, M. and BEN-Am, Y. (1990) Block of GABAs-activated K + conductance by kainate and quisqualate in rat CA3 hippocampal pyramidal neurones. Pfliigers Arch. 415, 471-478. SAITO, K., BARBER,R., Wu, J.-Y., MATSUDA,T., ROBERTS, E. and VAUGHN,J. E. (1974) Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proc. nam. Acad. Sci. U.S.A. 71, 269-273. SAKMANN,B., HAM1LL,O. P. and BORMANN,J. (1983) Patch clamp measurements of elementary chloride currents activated by the putative inhibitory transmitters GABA and glycine in mammalian spinal neurons, J. neural. Trans. 18(suppl.), 83-95. SATOU, M., MORI, K., TAZAWA, Y. and TAKAGI, S. F. (1982a) Two types of postsynaptic inhibition in pyriform cortex of the rabbit: fast and slow inhibitory postsynaptic potentials. J. Neurophysiol. 48, 1142-1156. SATOO, m., MORI, K., TAZAWA, Y. and TAKAGI, S. F. (1982b) Long-lasting disinhibition in pyriform cortex of the rabbit. J. Neurophysiol. 48, 1157-1163. SATOU, M., MORI, K., TAZAWA, Y. and TAKAGI, S. F. (1983a) Neuronal pathways for activation of inhibitory interneurones in pyriform cortex of the rabbit. J. Neurophysiol. 50, 74-88. SATOU, M., MOR1, K., TAZAWA, Y. and TAKAGL S. F. (1983b) Interneurones mediating fast postsynaptic inhibition in pyriform cortex of the rabbit. J. Neurophysiol. 50, 89-101. SCHLICHTER, R., DI~SARMENIEN,M., L1 VOLSl, G., DESAULLES,E. and FELTZ, P. (1987) Low concentrations of GABA reduce accommodation in primary afferent neurons by an action at GABA B receptors. Neuroscienee 20, 385-393. SCHNEIDER, S. P. and SCOTT, J. W. (1983) Orthodromic response properties of rat olfactory bulb mitral and tufted cells correlate with their projection patterns. J. Neurophysiol. 50, 358-378. SCHOEMAKER,H., BOLES,R. G., HORST, W. D. and YAMAMURA, H. I. (1983) Specific high-affinity binding sites for [3H]Ro 5-4864 in rat brain and kidney. J. Pharmac. exp. Ther. 225, 61-69. SCHOFmLD, P. R. (1989) The GABA A receptor: molecular biology reveals a complex picture. Trends Pharmac. Sci. 10, 476-478. SCHOFIELD, P. R., DARLISON, M. G., FUJITA, N., BURT, D. R., STEPHENSON,F. A., RODRIGUEZ,H., RHEE, L. M., RAMACHANDRAN, J., REALE, V., GLENCORSE, Y. A., SImBURG, P. H. and BARNARD, E. A. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328, 221-227. SCHOLFIELD,C. N. (1978a) A depolarizing inhibitory potential in neurones of the olfactory cortex in vitro. J. Physiol., Lond. 275, 547-557. SCHOLFIELD,C. N. (1978b) A barbiturate induced intensification of the inhibitory potential in slices of guinea-pig olfactory cortex. J. Physiol., Lond. 275, 559-566.

89

SCHOLFIELD, C. N. (1980a) Convulsants antagonize inhibition in the olfactory cortex slice. Naanyn-Schmiedebergs Arch. Pharmac. 314, 29-36. SCHOLEmLD, C. N. (1980b) Potentiation of inhibition by general anaesthetics in neurones of the olfactory cortex in vitro. Pfliigers Arch. 383, 249-255. SCHWARTZ, R. D., SKOLNICK,P., HOLLINGSWORTH, E. B. and PAUL, S. M. (1984) Barbiturate and picrotoxin-sensitive chloride effiux in rat cerebral synaptoneurosomes. FEBS Lett. 175, 193-196. SCHWARTZKROIN,P. A. and MATHERS,L. H. (1978) Physiological and morphological identification of a nonpyramidal hippocampal cell type. Brain Res. 157, 1-10. SEGAL, M. (1986) Properties of rat medial septal neurones recorded in vitro. J. Physiol., Lond. 379, 309-330. SEGAL, M. and BARKER, J. L. (1984a) Rat hippocampal neurons in culture: properties of GABA-activated e l - ion conductance. J. Neurophysiol. 51, 500-515. SEGAL, i . and BARKER, J. L. (1984b) Rat hippocampal neurons in culture: voltage-clamp analysis of inhibitory synaptic connections. J. Neurophysiol. 52, 469-487. SI~QU1ER,J. M., RICHAROS,J. G., MALHERBE,P., PRICE, G. W., MATHEWS,S. and MOHLER, H. (1988) Mapping the brain areas containing RNA homologous to cDNAs encoding the • and /~ subunits of the rat GABAA yaminobutyrate receptor. Proc. hath. Acad. Sci. U.S.A. 85, 7815-7819. SERESS, L. and RIBAK, C. E. (1983) GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons. Expl Brain Res. 50, 173-182. SHARIF, N. A. (1985) Multiple synaptic receptors for neuroactive amino acid transmitters--new vistas. Int. Rev. Neurobiol. 26, 85-150. SI-mRMAN, S. M. and KOCH, U (1986) The control of retinogeniculate transmission in the mammalian lateral geniculate nucleus. Expl Brain Res. 63, 1-20. SHIVERS,B. D., KILLISCH,I., SPRENGEL,R., SONTHEIMER,H., K6HLER, M., SCHOFIELD, P. R. and SEEBURG, P. H. (1989) Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Soc. Neurosci. Abstr. 15, 970. SmGHART, W. (1989) Multiplicity of GABAA-benzodiazepine receptors. Trends Pharmac. Sci. 10, 407-411. SIGEL, E. and BARNAR3, E. A. (1984) ~,-Aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. J. biol. Chem. 259, 7219-7223. SIMMONDS,M. A. and TURNER,J. P. (1987) Potentiators of responses to activation of y-aminobutyric acid (GABAA) receptors. Neuropharmacology 26, 923-930. SIVILOTTI, L. G. (1988) Pharmacological actions of neutral amino acids on synaptic transmission in the frog optic tectum. Ph.D. Thesis, University of London. SXVILOTTI, L. and NlSTRI, A. (1986) Biphasic effects of giycine on synaptic responses of the frog optic rectum in vitro. Neurosci. Lett. 66, 25-30. SIViLOTT1, L. and NXSTRI, A. (1988) Complex effects of baclofen on synaptic transmission of the frog optic tectum in vitro. Neurosci. Lett. 85, 249-254. SIVlLOTTLL. and NISTm, A. (1989) Pharmacology of a novel effect of y-aminobutyric acid on the frog optic tectum in vitro. Eur. J. Pharm. 164, 205-212. SKERR1TT, J. H. and JOHNSTON,G. A. R. (1983) Enhancement of GABA binding by benzodiazepines and related anxiolytics. Eur. J. Pharmac. 89, 193-198. SLOVlTER,R. S. and NiLAVER,G. (1987) Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide- and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat. J. comp. Neurol. 256, 42-60. SMITH,Y. and BOLAM,J. P. (1989) Neurons of the substantia nigra reticulata receive a dense GABA-containing input from the globus pallidus of the rat. Brain Res. 493, 160-167.

90


SNODGRASS,S. R. (1983) Receptors for amino acid transmitters. In: Handbook o f Psyehopharmacology, Vol. 17: Biochemical Studies o f C N S Receptors, pp. 167-239. Eds. L. L. IVERSEN, S. D. IVERSEN and S. H. SNYDER. Plenum Press: New York and London. SOLTESZ I., HABV, M., LERESCHE, N. and CRUNELLI, V. (1988) The GABAB antagonist phaclofen inhibits the late K+-dependent IPSP in cat and rat thalamic and hippocampal neurones. Brain Res. 448, 351-354. SOLTESZ, I., LIGHTOWLER, S., LERESCHE, N. and CRUNELLI, V. (1989a) On the properties and origin of the GABA e inhibitory postsynaptic potential recorded in morphologically identified projection cells of the cat dorsal lateral geniculate nucleus. Neuroscience 33, 23-33. SOLTESZ, 1., LIGHTOWLER, S., LERESCHE, N. and CRUNELLI, V. (1989b) Optic tract stimulation evokes GABAA but not GABA B IPSPs in the rat ventral lateral geniculate nucleus. Brain Res. 479, 49-55. SOMOGYI, P., SMITH, A. D., NUNZI, M. G., GORIO, A., TAKAGI, H. and Wu, J.-Y. (1983a) Glutamate decarboxylase immunoreactivity in the hippocampus of the cat: distribution of immunoreactive synaptic terminals with special reference to the axon initial segment of pyramidal neurons. J. Neurosci. 3, 1450-1468. SOMOGYI,P., FREUND, T. F., Wu, J.-Y. and SMITH,A. D. (1983b) The section-Golgi impregnation procedure. 2. Immunocytochemical demonstration of glutamate decarboxylase in Golgi impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat. Neuroscience 9, 475-490. SOMOGYI, P., TAKAGI, H., RICHARDS, J. G. and MOHLER, H. (1989) Subcellular localization of benzodiazepine/ GABA g receptors in the cerebellum of rat, cat and monkey using monoclonal antibodies. J. Neurosci. 9, 2197-2209. SOR1ANO, E. and FROTSCHER, M. (1989) A GABAergic axo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway. Brain Res. 503, 170-174. SPREAFICO, R., SCHMECHEL, D. E., ELLIS, L. C. JR and RUSTIONI, A. (1983) Cortical relay neurons and interneurons in the n. ventralis posterolateralis of cats: a horseradish peroxidase, electron microscopic, Golgi and immunocytochemical study. Neuroscience 9, 491509. SQUIRES, R. F. and BRAESTRUP, C. (1977) Benzodiazepine receptors in rat brain. Nature 266, 732-734. SQUIRES, R. F., CASlDA, J. E., RICHARDSON, M. and SAEDERUP, E. (1983) [35S]t-Butyl-bicyclophosphorothionate binds with high affinity to brain specific sites coupled to 7-aminobutyric acid-A and ion recognition sites. Molec. Pharmac. 23, 326-336. STEFANIS, C. and JASPER, H. (1964) Intracellular microelectrode studies of antidromic responses in cortical pyramidal tract neurons. J. Neurophysiol. 27, 828-854. STEPHENSON, F. A. (1988) Understanding the GABA A receptor: a chemically gated ion channel. Biochem. J. 249, 21 32. STEPHENSON, F. A. and DOLPHIN, A. C. (1989) GABA and glycine neurotransmission. Seminars in the Neurosciences, 1, 115 123. STERIADE, M. and DESCH~NES, M. (1984) The thalamus as a neuronal oscillator. Brain Res. Rev. $, 1-63. STERLING, P. and DAVIS, T. L. (1980) Neurons in cat lateral geniculate nucleus that concentrate exogenous [3H]-yaminobutyric acid (GABA). J. comp. Neurol. 192, 737-749. STEVENS, D. R., GALLAGHER, J. P. and SHINNICK-GALLAGHER, P. (1987) In vitro studies of the role of yaminobutyric acid in inhibition in the lateral septum of the rat. Synapse !, 184-190. STORM-MATmSEN, J. and OPSAHL, M. W. (1978) Aspartate and/or glutamate may be transmitters in hippocampal

efferents to septum and hypothalamus. Neurosci. Left. 9, 65-70. STORM-MATHISEN, J., LEKNES, A. K., BOP,E, A. T., VAALAND, J. L., EDMINSON, P., HANG, F. M. S. and OTTERSEN, O. P. (1983) First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517-520. STUDY, R. E. and BARKER, J. L. (1981) Diazepam and (-)pentobarbital: fluctuation analysis reveals different mechanisms for potentiation of y-aminobutyric acid responses in cultured central neurons. Proc. natn. Acad. Sci. U.S.A. 78, 7180-7184. SUZDAK,P. D., SCHWARTZ,R. D., SKOLNICK,P. and PAUL, S. P. (1986) Ethanol stimulates y-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc. natn. Acad. Sci. U.S.A. 83, 4071-4075. SYKES, T. C. F. and THOMSON, A. M. (1989) Sodium pentobarbitone enhances responses of thalamic relay neurones to GABA in rat brain slices. Br. d. Pharmac. 97, 1059-1066. TAFT, W. C. and DELORENZO, R. J. (1984) Micromolaraffinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations. Proc. natn. Acad. Sci. U.S.A. gl, 3118-3122. TAGUCHI, J., KURIYAMA,T., OHMOR1,Y. and KURIYAMA,K. (1989) lmmunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABA A receptor complex. Brain Res. 483, 395-401. TALLMAN,J. F., THOMAS,J. W. and GALLAGER,D. W. (1978) GABAergic modulation of benzodiazepine binding site sensitivity. Nature 274, 383-385. TEHRANI, M. H. J., VAIDYANATHASWAMY, R., VERKADE, J. G. and BARNES, M. (1986) Interaction of t-butylbicyclophosphorothionate with 7-aminobutyric acid-gated chloride channels in cultured central neurons. J. Neurochem. 46, 1542-1548. THALMANN, R. H. (1988) Blockade of a late inhibitory postsynaptic potential in hippocampal CA3 neurons in vitro reveals a late depolarizing potential that is augmented by pentobarbital. Neurosci. Lett. 95, 155-160. THALMANN, R. H. and AYALA, G. F. (1982) A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus. Neurosci. Lett. 29, 243-248. THALMANN, R. H. and HERSHKOWITZ, N. (1985) Some factors that influence the decrement in the response to GABA during its continuous iontophoretic application to hippocampal neurons. Brain Res. 342, 219-233. THAMPY, K. G. and BARNES, E. M. JR (1984) 7-Aminobutyric acid-gated channels in cultured neurons. J. biol. Chem. 259, 1753-1757. THOMAS, R. C. (1984) Experimental displacement of intracellular pH and the mechanism of its subsequent recovery. J. Physiol., Lond. 354, 3P-22P. THOMPSON, S. M. and G.KHWILER, B. H. (1989a) Activity-dependent disinhibition. I. Repetitive stimulation reduces IPSP driving force and conductance in the hippocampus in vitro. J. Neurophysiol. 61, 501-511. THOMPSON, S. M. and G~.HWlLER, B. H. (1989b)Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on Ect- in hippocampal CA3 neurons. J. Neurophysiol. 61,512-523. THOMPSON, S. M. and G~,HWlLER, B. H. (1989c) Activity-dependent disinhibition. III, Desensitization and GAB_AB receptor-mediated presynaptic inhibition in the hippocampus in vitro. J. Neurophysiol. 61, 524-533. THOMPSON, S. M., DEISZ, R. A. and PRINCE, D. A. (1988) Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons. J. Neurophysiol. 60, 105-124. THOMSON, A. M. (1988a) IPSPs evoked in thalamic neurones by stimulation of the reticularis nucleus. Neuroscienee 25, 491-502.

GABA RECEPTORMECHANISMS THOMSON, A. M. (1988b) Biphasic responses of thalamic neurones to ~-aminobutyric acid in isolated rat brain slices. Neuroscience 25, 503-512. TICKU, M. K. and MAKSAY, G. (1983) Convulsant/ depressant site of action at the allosteric benzodiazepineGABA receptor-ionophore complex. Life Sci. 33, 2363-2375. TICKU, M. K. and OLSEN, R. W. (1978) Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system. Life Sci. 22, 1643-1651. TlcKU, M. K. and OLSEN, R. W. (1979) Cage convulsant drugs inhibit dihydropicrotoxinin binding. Neuropharmacology 18, 315-318. TSENG, G. F. and HABERLY,L. B. (1988) Characterization of synaptically-mediated fast and slow inhibitory processes in pyriform cortex in an in vitro slice preparation. J. Neurophysiol. 1352-1376. TUNNICLIFF, G. and SMITH, J. A. (1981) Competitive inhibition of 7-aminobutyric acid receptor binding by N-2 hydroxy-ethylpiperazine-N-2-ethanesulfonic acid and related buffers. J. Neurochem. 36, 1122-1126. TURNER, A. J. and WmYrLE, S. R. (1983) Biochemical dissection of the y-aminobutyrate synapse. Biochem. J. 209, 29~,1. TURNER, D. M., RANSOM, R. W., YANG, J. S.-J. and OLSEN, R. W. (1989) Steroid anaesthetics and naturally occurring analogs modulate the ~,-aminobutyric acid receptor complex at a site distinct from barbiturates. J. Pharmac. exp. Ther. 248, 960-966. TWYMAN, R. E., ROGERS, C. J. and MACOONALD,R. L. (1989a) Pentobarbital and picrotoxin have reciprocal actions on single GABA A receptor channels. Neurosci. Lett. 96, 89-95. TWYMAN, R. E., ROGERS, C. J. and MACDONALD,R. L. (1989b) Differential regulation of y-aminobutyric acid receptor channels by diazepam and phenobarbital. Ann. Neurol. 25, 213-220. UCmMURA,N., HIGASHLH. and Nxsm, S. (1989) Membrane properties and synaptic responses of the guinea-pig nucleus accumbens neurons in vitro. J. Neurophysiol. 61, 769-779. UNNERSTALL, J. R., KUHAR, M. J., NmHOFF, D. L. and PALACIOS, J. M. (1981) Benzodiazepine receptors are coupled to a subpopulation of ~,-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study. J. Pharmac. exp. Ther. 218, 797-804. VANDERMAELEN,C. P. and KITAI, S. T. (1980) An intracellular analysis of synaptic potentials in rat neostriatum following stimulation of the cerebral cortex, thalamus and substantia nigra. Brain Res. Bull. 5, 725-733. VANDERMAELEN,C. e., BONDUKI, A. C. and KITAI, S. T. (1979) Excitation of caudate-putamen neurons following stimulation of the dorsal raphe nucleus in the rat. Brain Res. 175, 356-361. VERDOORN,T. A., DRAGUHN,A., SAKMANN,B., PRITCHETT, D. B. and SEEBURG,P. H. (1989) Human GABAA receptors assembled from different combinations of cloned subunits have different electrophysiological properties. Soc. Neurosci. Abstr. 15, 970. VERMA, A. and SNYDER, S. H. (1989) Peripheral type benzodiazepine receptors. A. Rev. Pharmac. Toxicol. 29, 307-322. VICINI, S., MIENVlLLE,J.-M. and COSTA, E. (1987) Actions of benzodiazepine and fl-carboline derivatives on yaminobutyric acid-activated Cl- channels recorded from membrane patches of neonatal rat cortical neurones in culture. J. Pharmac. exp. Ther. 243, 1195-1201. VINCENT,S. R., H6K~LT, T., SKmBOLL,L. R. and Wu, J.-Y. (1983) Hypothalamic 7-aminobutyric acid neurons project to the neocortex. Science 220, 1309-1311. WALAAS, I. and FONNUM, F. (1979) The effects of surgical and chemical lesions on neurotransmitter candidates in

91

the nucleus accumbens of the rat. Neuroscience 4, 209-216. WALDMEIER, P. C., WICKI, P., FELDTRAUER, J.-J. and BAUMANN, P. A. (1988) Potential involvement of a baclofen-sensitive autoreceptor in the modulation of the release of endogenous GABA from rat brain slices in vitro. Naunyn-Schmiedebergs Arch. Pharmac. 337, 289-295. WANG, M. Y. and DuN, N. J, (1990) Phaclofen-insensitive presynaptic inhibitory action of (+)-baclofen in neonatal rat motoneurones in vitro. Br. J. Pharmac. 99, 413-421. WEIss, D. S. (1988) Membrane potential modulates the activation of GABA-gated channels. J. Neurophysiol. 59, 514-527. WExss, D. S. and HABLITZ, J. J. (1984) Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex. Cell molec. Neurobiol. 4, 301-317. WEIss, D. S. and MAGLEBY,K. L. (1989) Gating scheme for single GABA-activated C1- channels determined from stability plots, dwell-time distributions, and adjacent-interval durations. J. Neurosci. 9, 1314-1324. WEIss, D. S., BARNES, E. M. and HABLITZ, J. J. (1988) Whole-cell and single-channel recordings of GABA-gated currents in cultured chick cerebral neurons. J. Neurophysiol. 59, 495-513. WERMAN, R., DAVIOO~, R. A, and Al,mSOy, M. H, (1968) Inhibitory actions of glycine on spinal neurons in the cat. J. Neurophysiol. 31, 81-95. WHITTLE,S. R. and TURNER,A. J. (1982) Differential effects of sedative and anticonvulsant barbiturates on specific [3H]GABA binding to membrane preparations from rat brain cortex. Biochem. Pharmac. 31, 2891-2895. WmKIN, G. P., CSmLAG,A., BALAZS,R., KINGSBURV,A. E., WILSON, J. E. and JOHNSON,A. L. (1981) Localization of high affinity [3H]glycine transport sites in the cerebellar cortex. Brain Res. 216, 11-33. WmLIAMS,M. and RISLEY,E. A. (1979) Enhancement of the binding of 3H diazepam to rat brain membranes in vitro by SQ 20009, a novel anxiolytic, 7-aminobutyric acid (GABA) and muscimol. Life Sci. 24, 833-842. WILLOW, M. and JOHNSTON,G. A. R. (1981) Dual action of pentobarbitone on GABA binding: role of binding site integrity. J. Neurochem. 37, 1291-1294. WILSON, C. J., CHANG, H. T. and KITAI, S. T. (1983) Disfacilitation and long-lasting inhibition of neostriatal neurons in the rat. Expl Brain Res. 51, 227-235. WLSE, S. P. (1985) Co-localization of GABA and neuropeptides in the cerebral cortex. Trends Neurosci. 8, 92-95. WOJTOWICZ,J. M., MARSHALL,K. C. and HENDELMAN,W. J. (1980) Electrophysiological and pharmacological studies of the inhibitory projection from the cerebellar cortex to the deep cerebellar nuclei in tissue culture. Neuroscience 3, 607-618. WONG, E. H. F., LEEB-LuNOSERG, L. M. F., TEICHBERG, V. I. and OLSEN, R. W. (1984) ~,-Aminobutyric acid activation of 36C1- flux in rat hippocampal slices and its potentiation by barbiturates. Brain Res. 303, 267-275. WOODSON,W., NI~CKA, L. and BEN-ARt,Y. (1989) Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study. J. comp. Neurol. 280, 254-271. YAMAMO'rO,C., YAMAMOTO,T. and IWAMA,K. (1963) The inhibitory system in the olfactory bulb studied by intracellular recording, J. Neurophysiol. 26, 403-415. YAMAMURA,H. I., MIMAKI,T., YAMAMURA,S. H., HORST, W. D., MOr,ELLL M., BAUTZ, G. and O'BRIEN, R. A. (1982) [3H]CL 218,872, a novel triazolopyridazine which labels the benzodiazepine receptor in rat brain. Eur. J. Pharmac. 77, 351-354. YANG, J. S.-J. and OLSEN, R. W. (1987a) ~-Aminobutyric acid receptor binding in fresh mouse brain membranes at

92

L. SIVILOTTIand A. NlSTRI

22 C: ligand-induced changes in affinity. Molec. Pharmac. 32, 266-277. YANG, J. S.-J. and OLSEN, R. W. (1987b) ~t-Aminobutyric acid receptor-regulated 36C1- flux in mouse cortical slices. J. Pharmac. exp. Ther. 241, 677-685. YOSmDA, M. and PRECHT, W. (1971) Monosynaptic inhibition of neurons of the substantia nigra by caudatonigral fibres. Brain Res. 32, 225-228.

YOUNG, W. S., N1EHOFF, D., KUHAR, M. J., BEER B. and LIPPA, A. S. (1981) Multiple benzodiazepine receptor localization by light microscopic radiohistochemistry. J. Pharmac. exp. Ther. 216, 425-430. ZUKIN, S. R., YOUNG, A. B. and SNYDER, S. H. (1974) Gamma-aminobutyric acid binding to receptor sites in the rat central nervous system. Proc. natn. Acad. Sci. U.S.A. 71, 4802-4807.

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