GABA Receptor Subtypes Research Articles

Modulation of the components of the rat dark-adapted electroretinogram by the three subtypes of GABA receptors

The separate components of the dark-adapted electroretinogram (ERG) are believed to reflect the electric activity of neurones in both the inner and the outer layers of the retina, although their precise origin still remains unclear. The purpose of this study was to examine whether selective blockage or stimulation of the different subtypes of GABA receptors might help further elucidate the cellular origin of the components of the dark-adapted ERG. The rat retina is of interest since the localization and physiology of GABA receptors in that retina have been examined in great detail. GABA agonists and antagonists, known to affect the responses of neurons in the inner plexiform layer, were injected into the vitreous of one eye while ERG responses evoked by flashes of white light were recorded. GABA and the GABAa agonist isoguvacine completely removed the oscillatory potentials (OPs) and reduced the amplitude of the a- and b-waves. TPMPA, a GABAC antagonist, reduced the a- and b-waves but had no significant effect on the OPs. Baclofen, a GABAb agonist, reduced the amplitude of the a- and b-waves, without having any effects on the amplitude of the OPs. The GABAb antagonist CGP35348 increased the amplitudes of the a- and b-wave without having an effect on the amplitudes of the OPs. The GABAb receptor ligands had significant and opposite effect on the latency of the OPs. These results indicate that retinal neurons, presumably a subpopulation of amacrine cells, that have GABAb receptors are not the source of the OPs of the ERG, although they may modulate these wavelets in some manner, while contributing to the generation of the dark-adapted a- and b-waves. OPs are modified by stimulation of GABAa receptors, and the a- and b-waves by stimulation of all GABA receptor subtypes.

Methodological approaches for the study of GABA(A) receptor pharmacology and functional responses.

Inhibitory GABA(A) receptor ion channels are the target for a wide range of clinically-used therapeutic agents. The complex structural diversity of these ligand-gated channels, revealed by molecular cloning studies, together with increasing requirements for higher-throughput functional assays in drug discovery, has led to the development of a wide range of techniques to examine GABA(A) receptor pharmacology and function. In the current article we review some of the methodologies which have contributed to the expansion of knowledge in this field. The techniques include: molecular approaches, immunoprecipitation, and immunopurification to study receptor assembly, structure, and functional expression; in situ hybridization, immunocytochemistry, and autoradiography to examine receptor distribution in native tissues; radioligand binding, site-directed mutagenesis, and electrophysiology to examine pharmacology and allosteric modulation; and patch clamp, ion flux, microphysiometry, and a variety of novel fluorescence-based technologies to examine ion-channel function. The use of gene targetting approaches in transgenic mice has also provided important insights into the role of specific GABA(A) receptor subtypes in vivo. The continuing evolution of novel technologies and assay approaches with appropriate sensitivity and resolution to measure subtle modulation of GABA(A) ion channels will facilitate ongoing investigation of the physiological functions of these important inhibitory receptors.

GABA(A) receptor subtype selective cognition enhancers.

Currently the treatment of Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI) is largely unrealised, with no preventive or curative therapies. The marketed acetylcholinesterase inhibitors (eg. donepezil, Aricept) are directed toward temporary symptomatic relief from impaired cognition, but have prominent adverse effects with minimal efficacy. In pursuit of novel cognition enhancers, the observation that classical benzodiazepines (BZ, eg. diazepam) are amnesic, coupled with the preservation of GABA(A) receptors in brain areas most affected by AD, highlighted the GABA(A) receptor as a potential therapeutic target. In contrast to the amnesic BZ agonists, the BZ inverse agonists (eg. DMCM) which attenuate GABA(A) receptor function, have been shown to improve performance in animal models of learning and memory. Unfortunately, such non-selective ligands also induce anxiety and convulsions. More recently, novel ligands have been developed (eg. 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one) that demonstrate binding selectivity and high inverse agonism for the alpha5 GABA(A) receptor subtype, which is preferentially located in the hippocampus, a region of the brain associated with learning and memory. Pre-clinical results are encouraging, since these alpha5 selective inverse agonists enhance memory in animal models, such as spatial learning in the Morris water-maze, but are devoid of the adverse effects associated with activity at other GABA(A) receptor subtypes in other brain regions. If the efficacy and safety profiles of alpha5 inverse agonists in humans prove to be similar to those seen in pre-clinical studies, these compounds would offer significant benefit to AD and MCI patients.

Anxioselective compounds acting at the GABA(A) receptor benzodiazepine binding site.

With the exception of obsessive compulsive disorder, benzodiazepines (BZs) remain a major first line treatment for anxiety disorders. However, as well as being anxiolytic, BZs also cause sedation acutely, related to the fact that BZs are also used as hypnotics, and chronically may have abuse potential as well as cause physical dependence which manifests itself as the demonstration of a number of adverse events upon discontinuation. The molecular mechanisms of BZs are now well defined in that they enhance the actions of the inhibitory neurotransmitter GABA by binding to a specific recognition site on GABA(A) receptors containing alpha1, alpha2, alpha3 and alpha5 subunits. Compounds that bind at this modulatory site and enhance the inhibitory actions of GABA are classified as agonists, those that decrease the actions of GABA are termed inverse agonists whereas compounds which bind but have no effect on GABA inhibition are termed antagonists. The clinically used BZs are full agonists and between the opposite ends of the spectrum, i.e. full agonist and full inverse agonist, are a range of compounds with differing degrees of efficacy, such as partial agonists and partial inverse agonists. Attempts have been made to develop compounds which are anxioselective in that they retain the anxiolytic properties of the full agonist BZs but have reduced sedation and dependence (withdrawal) liabilities. Such compounds may interact with all four (i.e. alpha1-, alpha2-, alpha3- and alpha5-containing) GABA(A) receptor subtypes and have partial rather than full agonist efficacies. Examples of nonselective partial agonists include bretazenil, imidazenil, FG 8205, abecarnil, NS 2710, pagoclone, RWJ-51204 and (S)-desmethylzopiclone. Alternatively, a compound might have comparable binding affinity but different efficacies at the various subtypes, thereby preferentially exerting its effects at subtypes thought to be associated with anxiety (alpha2- and/or alpha3-containing receptors) rather than the subtype associated with sedation (alpha1-containing receptors). Examples of efficacy selective compounds include L-838417, NGD 91-3 and SL651498. For each compound, preclinical and where available clinical data will be reviewed. Emerging themes include the lack of definitive intrinsic efficacy data for certain compounds (e.g. abecarnil, ocinaplon, pagoclone) and the difficulty in translating robust anxiolysis and a separation between anxiolytic and sedative doses of non-selective partial agonists in preclinical species into consistent clinical benefit in man (e.g. bretazenil, abecarnil, pagoclone). With respect to efficacy selective compounds, NGD 91-3 was not anxiolytic in man but in the absence of efficacy data, these results are difficult to interpret. Nevertheless, efficacy selective compounds represent a novel approach to targeting specific subtypes of the GABA(A) receptor, the ultimate test of which will be evaluation in the clinic.

Validation of GABA(A) receptor subtypes as potential drug targets by using genetically modified mice.

A key issue for drug discovery in the post genomic era is target validation. This is particularly important when considering the CNS, where currently the majority of drug targets are neurotransmitter receptors that are known to exist as multi-gene families. The GABAergic system, which is the major inhibitory neurotransmitter system in the CNS, is no exception in that respect. The GABA(A) receptors, which are the site of action of a number of clinically used drugs such as benzodiazepines and barbiturates, exist in a large gene family. Existing drugs mediating their effects through the GABA(A) receptor are generally non-selective, i.e. will act at several subtypes of that receptor family. Thus, if we are both to refine existing therapeutic approaches, and develop novel approaches, a key question is to define which subtype(s) of the GABA(A) receptor family we should target; which will mediate the beneficial effects of a drug, and which could be responsible for unwanted side effects? One of the tools, which has been developed over the last decade to elucidate the function of a given gene, is the generation and analysis of gene-targeted mice. This review will summarize progress on identifying individual GABA(A) receptor subtypes as potential drug targets by using genetically modified mice.

Hindquarters vasoconstriction through central GABA(B) receptors in conscious rats.

The exogenous application of GABA into the cisterna magna of the freely moving rat decreases hindquarters vascular tone as well as arterial pressure. GABA could influence GABA receptor subtypes A, B or C. However, the hindquarters vascular response to the stimulation of each receptor subtype has not yet been investigated. The present study therefore characterized the response to the GABA(B) receptor agonist baclofen injected into the cisterna magna of the conscious rat. Intracisternally injected baclofen induced long-lasting increases in hindquarters vascular resistance and arterial pressure in a dose-dependent manner. Both actions induced by baclofen were completely blocked by a prior intracisternal injection with the GABA(B) receptor antagonist CGP 35348 (p-[3-aminopropyl]-p-diethoxymethylphosphinic acid), and systemically by ganglionic blockade. These actions of baclofen were also abolished centrally by sodium pentobarbital anaesthesia. The results suggest that GABA(B) receptor stimulation via the cisterna magna induced hindquarters vasoconstriction, probably due to central disinhibition of sympathetic activity.

GABA Receptors Containing Rdl Subunits Mediate Fast Inhibitory Synaptic Transmission inDrosophilaNeurons

GABAergic inhibition in Drosophila, as in other insects and mammals, is important for regulation of activity in the CNS. However, the functional properties of synaptic GABA receptors in Drosophila have not been described. Here, we report that spontaneous GABAergic postsynaptic currents (sPSCs) in cultured embryonic Drosophila neurons are mediated by picrotoxin-sensitive chloride-conducting receptors. A rapid increase in spontaneous firing in response to bath application of picrotoxin demonstrates that these GABA receptors mediate inhibition in the neuronal networks formed in culture. Many of the spontaneous GABAergic synaptic currents are sodium action potential independent [miniature IPSCs (mIPSCs)] but are regulated by external calcium levels. The large variation in mIPSC frequency, amplitude, and kinetics properties between neurons suggests heterogeneity in GABA receptor number, location, and/or subtype. A decrease in the mean mIPSC decay time constant between 2 and 5 d, in the absence of a correlated change in rise time, demonstrates that the functional properties of the synaptic GABA receptors are regulated during maturation in vitro. Finally, neurons from the GABA receptor subunit mutant Rdl exhibit reduced sensitivity to picrotoxin blockade of the mIPSCs and resistance to picrotoxin-induced increases in spontaneous firing frequency. This demonstrates that Rdl containing GABA receptors play a direct role in mediating synaptic inhibition in Drosophila neural circuits formed in culture.

Lack of intersite GABA receptor subtype antagonist effects upon μ opioid receptor agonist-induced feeding elicited from either the ventral tegmental area or nucleus accumbens shell in rats

Pretreatment with the GABA A receptor antagonist, bicuculline or the GABA B receptor antagonist, saclofen, into the nucleus accumbens (Nacc) shell, respectively, potentiates and reduces feeding elicited by the μ opioid agonist, [ d-Ala 2, Nme 4, Gly-ol 5]-enkephalin (DAMGO), administered into the same site. DAMGO-induced feeding elicited from the ventral tegmental area (VTA) region is significantly reduced by pretreatment with saclofen into the same site indicating local GABA mediation of opioid-induced feeding in each site. Given the neuroanatomical and functional connections between the two sites, the present study evaluated the dose-dependent actions of bicuculline and saclofen pretreatment in one site upon DAMGO-induced feeding elicited from the second site. Pretreatment of either bicuculline (7.5–75 ng) or saclofen (1.5–10 μg) into the Nacc shell failed to alter the time course or magnitude of DAMGO-induced feeding elicited from the VTA region. DAMGO-induced feeding elicited from the Nacc shell was unaffected by VTA region pretreatment with either bicuculline (7.5–75 ng) or saclofen (1.5–5 μg). A higher (10 μg) saclofen dose prevented significant DAMGO-induced feeding after 1 and 4 h. Thus, although GABA receptor subtype antagonists are capable of differentially modulating DAMGO-induced feeding when both drugs are applied locally in either the VTA region or the Nacc shell, it appears that any effects between the VTA region and the Nacc shell in modulating DAMGO-induced feeding do not depend upon a GABAergic synapse in the other site.

Identification of a novel, selective GABA(A) alpha5 receptor inverse agonist which enhances cognition.

In pursuit of a GABA(A) alpha5-subtype-selective inverse agonist to enhance cognition, a series of 6,7-dihydro-2-benzothiophen-4(5H)-ones has been identified as a novel class of GABA(A) receptor ligands. These thiophenes have higher binding affinity for the GABA(A) alpha5 receptor subtype compared to the GABA(A) alpha1, alpha2, and alpha3 subtypes, and several analogues exhibit high GABA(A) alpha5 receptor inverse agonism. 6,6-Dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (43) has been identified as a full inverse agonist at the GABA(A) alpha5 receptor and is functionally selective over the other major GABA(A) receptor subtypes. 43 readily penetrates into the CNS to give selective occupancy of GABA(A) alpha5 receptors. In addition, 43 enhances cognitive performance in rats in the delayed 'matching-to-place' Morris water maze test-a hippocampal-dependent memory task-without the convulsant or proconvulsant activity associated with nonselective, GABA(A) receptor inverse agonists.

Localization of GABAA receptor subunits alpha 1, alpha 3, beta 1, beta 2/3, gamma 1, and gamma 2 in the salamander retina.

Electrophysiological studies have demonstrated that gamma-aminobutyric acid receptors type A (GABA(A)) mediate important information processing in the retinas of salamander and other vertebrates. The pharmacology and physiology of GABA(A) receptors depend on their subunit composition. We studied the localization of GABA(A) receptor subunit isoforms alpha(1), alpha(3), beta(1), beta(2/3) (antibody BD-17 and 62-3G1), gamma(1), and gamma(2) in salamander retina with immunocytochemical methods. All three beta-subunit antibodies labeled similarly in the outer retina, especially the inner segments and synaptic terminals of rod photoreceptors (identified with protein kinase C). Somatic labeling was observed in cell bodies of some horizontal cells, bipolar cells, amacrine cells, and cells in the ganglion cell layer (GCL). Puncta were present throughout the inner plexiform layer (IPL) for beta(1) and 62-3G1, but not for BD-17. alpha(1)-immunoreactivity (IR) stained a population of presumed OFF rod-dominated bipolar cells, including dendrites, soma, and axon terminals in the distal IPL. A subtype of GABAergic amacrine cell also expressed alpha(1)-IR, with puncta sparsely distributed at the distal and proximal margins of the IPL. Both the OPL and IPL were labeled throughout for alpha(3)-IR, as opposed to the narrow distribution of alpha(1)-IR in the IPL, suggesting that the two alpha-subunits are localized at different synaptic sites. Punctate gamma(1)-IR was observed in the OPL and IPL, whereas gamma(2) was most prominent in cone photoreceptors (identified with calbindin), including the terminal telodendria, in cell bodies of some horizontal cells, amacrine cells, cells in the GCL, and less intensely in the IPL. In addition, several subunits were present in Müller cells. The differential labeling suggests the existence of GABA(A) receptor subtypes with different subunit compositions that mediate multiple GABAergic functions in salamander retina.

Role of GABA A and GABA B receptors in GABA-induced inhibition of rat red nucleus neurons

We investigated GABA receptor subtypes mediating GABA-induced inhibition of red nucleus (RN) neuronal firing recorded extracellularly from anaesthetized rats. GABA response was mimicked by the GABA A agonists muscimol and isoguvacine in all cases and was partially blocked by the GABA A antagonist bicuculline. The GABA B agonist baclofen induced a long-lasting inhibition in 84% of cells. Neurons responding to either GABA A or GABA B agonists were equally distributed within the RN. The GABA C receptor agonist cis-amino-crotonic acid (CACA) did not modify RN neuronal firing; at high doses CACA occasionally induced inhibition abolished by bicuculline and thus mediated by GABA A receptors. We conclude that the inhibitory effects of GABA in the RN are mediated by both GABA A and GABA B receptors, whereas GABA C receptors are not involved.

SL651498, a GABAA receptor agonist with subtype-selective efficacy, as a potential treatment for generalized anxiety disorder and muscle spasms.

SL651498 (6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one) was identified as a drug development candidate from a research program designed to discover subtype-selective GABA(A) receptor agonists for the treatment of generalized anxiety disorder and muscle spasms. The drug displays high affinity for rat native GABA(A) receptors containing alpha(1) (K(i) = 6.8 nM) and alpha(2) (K(i) = 12.3 nM) subunits, and weaker affinity for alpha5-containing GABA(A) receptors (K(i) = 117 nM). Studies on recombinant rat GABA(A) receptors confirm these findings and indicate intermediate affinity for the alpha(3)beta(2)gamma(2) subtype. SL651498 behaves as a full agonist at recombinant rat GABA(A) receptors containing alpha(2) and alpha(3) subunits, and as a partial agonist at recombinant GABA(A) receptors expressing alpha(1) and alpha(5) subunits. SL651498 produced anxiolytic-like and skeletal muscle relaxant effects qualitatively similar to those of benzodiazepines (BZs) [minimal effective dose (MED): 1 to 10 mg/kg, i.p. and 3 to 10 mg/kg, p.o.]. However, unlike these latter drugs, SL651498 induced muscle weakness, ataxia or sedation at doses much higher than those having anxiolytic-like activity (MED: 30 to 100 mg/kg, i.p. or p.o.). Moreover, in contrast to BZs, SL651498 did not produce tolerance to its anticonvulsant activity or physical dependence. It was much less active than BZs in potentiating the depressant effects of ethanol or impairing cognitive processes in rodents. The differential profile of SL651498 as compared to BZs may be related to its selective efficacy at the alpha(2)- and alpha(3)-containing GABA(A) receptors. This suggests that selectively targeting GABA(A) receptor subtypes can lead to drugs with increased clinical specificity. SL651498 represents a promising alternative to agents currently used for the treatment of anxiety disorders and muscle spasms without the major side effects seen with classical BZs.

Deletion of GABAA receptor alpha 1 subunit-containing receptors alters responses to ethanol and other anesthetics.

GABA(A) receptors have been implicated in mediating several acute effects of ethanol including anxiolysis, ataxia, sedation/hypnosis, and anticonvulsant activity. Ethanol sensitivity of neurons has been associated with expression of alpha1 subunit-containing receptors. The objective of this study was to determine the contribution of alpha1 subunit containing receptors to ethanol responses in comparison to neurosteroids and other anesthetics using GABA(A) receptor alpha1 subunit knockout mice. Deletion of alpha1 subunit-containing receptors did not alter the anxiolytic, ataxic, anticonvulsant, or hypnotic effects of ethanol or acute functional tolerance to ethanol but did increase sensitivity to the locomotor-stimulating effects of ethanol. The ability of ethanol to potentiate muscimol-stimulated chloride uptake and ethanol clearance was also not altered following alpha1 subunit deletion. The anticonvulsant and hypnotic effects of neurosteroids as well as their potentiating effect on GABA-mediated Cl(-) uptake were unaltered in alpha1(-/-) mice. The hypnotic effect of pentobarbital, etomidate, and midazolam were reduced, whereas the effect of ketamine was enhanced in alpha1(-/-) mice. Thus, GABA(A) receptor alpha1 subunit-containing receptors appear to influence the motor-stimulating effect of ethanol and the sedative/hypnotic effects of some anesthetics, but not ethanol. These receptors do not appear to be necessary for most ethanol responses, suggesting involvement of other GABA(A) receptor subtypes or other targets altogether.

Mutational analysis of molecular requirements for the actions of general anaesthetics at the γ-aminobutyric acidA receptor subtype, α1β2γ2

BackgroundAmino acids in the β subunit contribute to the action of general anaesthetics on GABAA receptors. We have now characterized the phenotypic effect of two β subunit mutations in the most abundant GABAA receptor subtype, α1β2γ2.ResultsThe β2(N265M) mutation in M2 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, while the direct actions of propofol, etomidate and alphaxalone were impaired. The β2(M286W) mutation in M3 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, whereas the direct action of propofol and etomidate, but not of alphaxalone, was impaired.ConclusionsWe found that the actions of general anaesthetics at α1β2(N265M)γ2 and α1β2(M286W)γ2 GABAA receptors are similar to those previously observed at α2β3(N265M)γ2 and α2β3(M286W)γ2 GABAA recpetors, respectively, with the notable exceptions that the direct action of propofol was decreased in α1β2(M286W)γ2 receptors but indistinguishable form wild type in α2β3(M286W)γ2 receptors and that the direct action of alphaxalone was decreased in α1β2(N265M)γ2 but not α2β3(N265M)γ2 receptors and indistinguishable form wild type in α1β2(M286W)γ2 receptors but increased in α2β3(M286W)γ2 receptors. Thus, selected phenotypic consequences of these two mutations are GABAA receptor subtype-specific.

Modulation of GABA(A) receptor function by nonhalogenated alkane anesthetics: the effects on agonist enhancement, direct activation, and inhibition.

At clinically relevant concentrations, ethers, alcohols, and halogenated alkanes enhance agonist action on the gamma-aminobutyric acid(A) (GABA(A)) receptor, whereas nonhalogenated alkanes do not. Many anesthetics also directly activate and/or inhibit GABA(A) receptors, actions that may produce important behavioral effects; although, the effects of nonhalogenated alkane anesthetics on GABA(A) receptor direct activation and inhibition have not been studied. In this study, we assessed the abilities of two representative nonhalogenated alkanes, cyclopropane and butane, to enhance agonist action, directly activate, and inhibit currents mediated by expressed alpha(1)beta(2)gamma(2L) GABA(A) receptors using electrophysiological techniques. Our studies reveal that cyclopro- pane and butane enhance agonist action on the GABA(A) receptor at concentrations that exceed those required to produce anesthesia. Neither nonhalogenated alkane directly activated nor inhibited GABA(A) receptors, even at concentrations that approach their aqueous saturated solubilities. These results strongly suggest that the behavioral actions of nonhalogenated alkane anesthetics do not result from their abilities to enhance agonist actions, directly activate, or inhibit alpha(1)beta(2)gamma(2L) GABA(A) receptors and are consistent with the hypothesis that electrostatic interactions between anesthetics and their protein binding sites modulate GABA(A) receptor potency. When normalized to either their in vivo anesthetic potencies or hydrophobicities, cyclopropane and butane are 1-1.5 orders of magnitude less potent enhancers of agonist action on alpha(1beta2gamma2L) GABA(A) receptors than isoflurane. Additionally, cyclopropane and butane fail to directly activate or inhibit receptors, even at near aqueous saturating concentrations. Thus, it is unlikely that either enhancement or inhibition of the most common GABA(A) receptor subtype in the brain accounts for the behavioral activities of cyclopropane and butane.

Synthesis and biological evaluation of 7,8,9,10-tetrahydroimidazo[1,2-c]pyrido[3,4-e]pyrimdin-5(6H)-ones as functionally selective ligands of the benzodiazepine receptor site on the GABA(A) receptor.

Benzodiazepines are allosteric modulators of the GABA(A) receptor. The traditionally prescribed benzodiazepines are nonselective and suffer from numerous side effects. Upon the identification of receptor subtypes, we set out to discover selective agents with the anticipation that these agents would have superior therapeutic potential. Herein, we describe the synthesis and biological evaluation of substituted 7,8,9,10-tetrahydroimidazo[1,2-c]pyrido[3,4-e]pyrimidin-5(6H)-ones and disclose that these compounds exhibit functional selectivity at the benzodiazepine receptor of GABA(A) receptor subtypes. The alpha(2)/alpha(3)-selective partial agonist 42 exhibited potent in vivo activity.

Functional characterization of GABA(A) receptors in neonatal hypothalamic brain slice.

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.

Pathophysiological aspects of diversity in neuronal inhibition: a new benzodiazepine pharmacology

Inhibitory interneurons in the brain provide the balance to excitatory signaling. On the basis of brain imaging and human genetics, a deficit in GABAergic inhibition (GABA, γ-aminobuiyric acid) has been identified as contributing to the pathophysiology of anxiety disorders, epilepsy, and schizophrenia. Therapeutically, GABA(A) receptors play a major role as targets for benzodiazepine drugs. The therapeutic relevance of the multitude of structurally diverse GABA(A) receptor subtypes has only recently been identified. α(1)-GABA(A) receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection of these drugs, whereas α(2)-GABA(A) receptors, but not α(3)-GABA(A) receptors, mediate anxiolysis. Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the upcoming subtype-selective drugs. For instance, anxiolytics devoid of drowsiness and sedation promise more sophisticated interventions in anxiety disorders. A new pharmacology of the benzodiazepine site is on the horizon.

Subunit composition, distribution and function of GABA(A) receptor subtypes.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. Depending on their subunit composition, these receptors exhibit distinct pharmacological and electrophysiological properties. Recent studies on recombinant and native GABA(A) receptors suggest the existence of far more receptor subtypes than previously assumed. Thus, receptors composed of one, two, three, four, or five different subunits might exist in the brain. Studies on the regional, cellular and subcellular distribution of GABA(A) receptor subunits, and on the co-localization of these subunits at the light and electron microscopic level for the first time provide information on the distribution of GABA(A) receptor subtypes in the brain. These studies will have to be complemented by electrophysiological and pharmacological studies on the respective recombinant and native receptors to finally identify the receptor subtypes present in the brain. The distinct cellular and subcellular location of individual receptor subtypes suggests that they exhibit specific functions in the brain that can be selectively modulated by subtype specific drugs. This conclusion is supported by the recent demonstration that different GABA(A) receptor subtypes mediate different effects of benzodiazepines. Together, these results should cause a revival of GABA(A) receptor research and strongly stimulate the development of drugs with a higher selectivity for alpha2-, alpha3-, or alpha5-subunit-containing receptor subtypes. Such drugs might exhibit quite selective clinical effects.

Medicinal Chemistry and Molecular Pharmacology of GABA-C Receptors

GABA(C) receptors belong to the nicotinicoid superfamily of ionotropic receptors that include nicotinic acetylcholine receptors, bicuculline-sensitive GABA(A) receptors, strychnine-sensitive glycine receptors and 5HT3 serotonin receptors. The GABA(C) receptor concept arose from medicinal chemical studies of a conformationally restricted analog of GABA. Receptors matching the predicted properties of GABA(C) receptors were cloned from the retina. Post cloning studies revealed the unique physiology and pharmacology of these relatively simple homomeric receptors. Three subtypes of GABA(C) receptors have been cloned from mammalian sources and pharmacological differences between the rho1, rho2 and rho3 GABA(C) receptors have been described. There is evidence for functional GABA(C) receptors in the retina, spinal cord, superior colliculus, pituitary and the gut and their involvement in vision, aspects of memory and sleep-waking behaviour. This review concentrates on the medicinal chemistry and molecular pharmacology of GABA(C) receptor subtypes emphasising possible new investigational tools with which to investigate further GABA(C) receptor function. The most useful currently available ligands that show some GABA(C) receptor subtype selectivity are TPMPA, P4PMA, imidazole-4-acetic acid, 2-methyl-TACA and (+/-)-TAMP.