Gamma-aminobutyric Acid Receptor Complex Research Articles

Effects of Methyl and Isopropyl N‐methylanthranilates from Choisya ternata Kunth (Rutaceae) on Experimental Anxiety and Depression in Mice

Choisya ternata Kunth (Rutaceae) is a plant species used in Mexican folk medicine for its antispasmodic and simulative properties. Recently, we identified a new alkaloid, isopropyl N-methylanthranilate, and a related one, methyl N-methylanthranilate, from the essential oil of this species and have proven them to possess antinociceptive activity even at 0.3 mg/kg. In the present study, anxiolytic and antidepressant effects of the two compounds have been studied in open field, horizontal wire, light/dark, forced swimming and tail suspension tests, as well as the effect on the onset and duration of diazepam-induced sleep in BALB/c mice. The volatile alkaloids (50-200 mg/kg, administered intraperitoneally), without having a muscle relaxant effect, caused a significant increase in the time the animals spent in an unsecured and putatively dangerous area when compared with the control group but had no effect on the number of crossings between the light/dark compartments. In addition to this anxiolytic activity, a significantly antidepressant-like effect was apparent at all tested doses, which was not due to an increase in locomotive activity. The anthranilates administered on their own did not induce sleep in mice but significantly prolonged the diazepam-induced sleep, in a dose-dependent way, suggesting an interaction with the gamma-aminobutyric acid receptor complex.

Decreased GABAA receptors and benzodiazepine binding sites in the anterior cingulate cortex in autism

The anterior cingulate cortex (ACC; BA 24) via its extensive limbic and high order association cortical connectivity to prefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio-emotional behavior. Multiple lines of evidence, including genetic and imaging studies, suggest that the ACC and gamma-amino-butyric acid (GABA) system may be affected in autism. The benzodiazepine binding site on the GABA(A) receptor complex is an important target for pharmacotherapy and has important clinical implications. The present multiple-concentration ligand-binding study utilized (3)H-muscimol and (3)H-flunitrazepam to determine the number (B(max)), binding affinity (K(d)), and distribution of GABA(A) receptors and benzodiazepine binding sites, respectively, in the ACC in adult autistic and control cases. Compared to controls, the autistic group had significant decreases in the mean density of GABA(A) receptors in the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine binding sites in the supragranular (28.9%) and infragranular (16.4%) lamina [corrected]. These findings suggest that in the autistic group this downregulation of both benzodiazepine sites and GABA(A) receptors in the ACC may be the result of increased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons as well as their output to key limbic cortical targets. Such disturbances likely underlie the core alterations in socio-emotional behaviors in autism.

Neurosteroids: synthesis and functions in the central and peripheral nervous systems.

Some steroids are synthesized within the central and peripheral nervous systems, mostly by glial cells. These are known as neurosteroids. In the brain, neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3 alpha,5 alpha-reduced metabolite 3 alpha,5 alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phophodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.

Pregnenolone sulfate enhances neurogenesis and PSA-NCAM in young and aged hippocampus

Age-dependent cognitive impairments have been correlated with functional and structural modifications in the hippocampal formation. In particular, the brain endogenous steroid pregnenolone-sulfate (Preg-S) is a cognitive enhancer whose hippocampal levels have been linked physiologically to cognitive performance in senescent animals. However, the mechanism of its actions remains unknown. Because neurogenesis is sensitive to hormonal influences, we examined the effect of Preg-S on neurogenesis, a novel form of plasticity, in young and old rats. We demonstrate that in vivo infusion of Preg-S stimulates neurogenesis and the expression of the polysialylated forms of NCAM, PSA-NCAM, in the dentate gyrus of 3- and 20-month-old rats. These influences on hippocampal plasticity are mediated by the modulation of the gamma-aminobutyric acid receptor complex A (GABA A) receptors present on hippocampal neuroblasts. In vitro, Preg-S stimulates the division of adult-derived spheres suggesting a direct influence on progenitors. These data provide evidence that neurosteroids represent one of the local secreted signals controlling hippocampal neurogenesis. Thus, therapies which stimulate neurosteroidogenesis could preserve hippocampal plasticity and prevent the appearance of age-related cognitive disturbances.

Modifying quinolone antibiotics yields new anxiolytics.

Patients taking fluoroquinolone antibiotics such as norfloxacin exhibit a low incidence of convulsions and anxiety. These side effects probably result from antagonism of the neurotransmitter gamma-aminobutyric acid (GABA) at the brain GABA(A) receptor complex (GRC). Modification of norfloxacin yields molecules such as compound 4 that potentiate GABA action with alpha(2) subunit selectivity. Compound 4 is anxiolytic but does not cause sedation, and may represent a new class of ligands that have anxiolytic activity without sedative liability.

Social and neural determinants of aggressive behavior: pharmacotherapeutic targets at serotonin, dopamine and gamma-aminobutyric acid systems.

Aggressive outbursts that result in harm and injury present a major problem for the public health and criminal justice systems, but there are no adequate treatment options. Obstacles at the level of social policy, institutional regulation, and scientific strategy in developing animal models continue to impede the development of specific anti-aggressive agents for emergency and long-term treatments. To be more relevant to the clinical situation, preclinical aggression research has begun to focus on the neurobiological determinants of escalated aggressive behavior that exceeds species-typical patterns. It is the goal of this review to examine novel pharmacological and molecular tools that target the neural mechanisms for different kinds of aggressive behavior more selectively than previously possible and to outline potential pharmacotherapeutic options. (1) The preclinical focus on the behavioral characteristics and determinants of intense aggression promises to be most relevant to the clinical distinction between the proposed impulsive-reactive-hostile-affective subtypes of human aggression and the controlled-proactive-instrumental-predatory subtypes of aggression. The neural circuits for many types of human and animal aggression critically involve serotonin, dopamine and gamma-aminobutyric acid (GABA) and specific receptor subtypes. (2) The dynamic changes in frontal cortical serotonin that are triggered by engaging in aggressive behavior imply that serotonergic drug effects are largely determined by the functional state of the receptors at the time of drug treatment. Of the numerous 5-HT receptors currently identified, the 5-HT(1B) receptors offer a promising target for reducing impulsive aggressive behavior, particularly if the action can be limited to sites in the central nervous system. (3) Aggressive confrontations are salient stressors, both for the aggressor as well as the victim of aggression, that are accompanied by activation of the mesocorticolimbic but not the striatal dopamine system. Dopaminergic manipulations, particularly targeting the D(2) receptor family, can influence aggressive behavior in animals and human patients, suggesting that mesocorticolimbic dopamine may have important enabling or permissive functions. (4) GABA is critical in the neurochemical control of aggressive behavior as evidenced by studies that directly modify GABAergic neurotransmission and neurochemical studies that correlate GABA measurements with aggressive behavioral responses in several animal species. The GABA(A) receptor complex is a mechanism through which certain benzodiazepines and alcohol enhance and inhibit aggressive behaviors. Social and pharmacological experiences decisively determine the effects of GABAergic positive modulators on aggression.

Ammonia, the GABA neurotransmitter system, and hepatic encephalopathy.

There appears to be a consensus that hepatic encephalopathy (HE) is a metabolic encephalopathy with a multifactorial pathogenesis. One of the factors considered to be important in the pathogenesis of HE is ammonia. However, the mechanisms by which ammonia contributes to the manifestations of HE remain poorly defined. Ammonia could be more definitively implicated in the pathogenesis of HE if its effects can be shown to lead to an enhancement of inhibitory neurotransmission. In this context the effects of ammonia on the GABA (gamma-aminobutyric acid) neurotransmitter system may be relevant. Ammonia, at the modestly increased concentrations that commonly occur in precoma HE (0.15 mM-0.75 mM), has been shown to increase GABA-induced chloride current in cultured neurons, probably by modifying the affinity of the GABA(A) receptor for GABA. Comparable ammonia concentrations also enhanced synergistically the binding of a GABA agonist and a benzodiazepine (BZ) agonist to the GABA(A) receptor complex, phenomena which would enhance the neuroinhibitory effects of these ligands. Also, GABA increased the potency of ammonia-induced enhancement of the binding of a BZ agonist to the GABA(A) receptor complex, and brain levels of BZ agonists are elevated in liver failure. In addition, ammonia has been shown to inhibit astrocytic uptake of GABA by 30%-50%, an effect which would increase the synaptic availability of GABA at GABA(A) receptors. Furthermore, increased ammonia concentrations upregulate the peripheral-type benzodiazepine receptor in the outer membrane of astroglial mitochondria, thereby enhancing astrocytic mitochondrial synthesis and release of neurosteroids. Some neurosteroids, for example tetrahydroprogesterone (THP) and tetrahydrodeoxycorticosterone (THDOC), are potent agonists of the GABA(A) receptor complex, on which there are specific binding sites for neurosteroids, that are distinct from those for BZs and barbiturates. Tetrahydroprogesterone and tetrahydrodeoxycorticosterone levels were found to be increased in a mouse model of acute liver failure, and, when THP or THDOC was injected into normal mice, sedation and Alzheimer type II astrocytic changes in the cortex, striatum, and hypothalmus were induced. Each of these direct or indirect effects of ammonia on the GABA neurotransmitter system has the potential of increasing inhibitory neurotransmission, and, hence, contributing to the manifestations of HE.

Nitrous oxide and xenon increase the efficacy of GABA at recombinant mammalian GABA(A) receptors.

We investigated the interactions between recombinant gamma-aminobutyric acid receptor complex (GABA(A)R) and nitrous oxide (N(2)O) or xenon (Xe). Human embryonic kidney cells (HEK 293) were transfected with rat cDNA for alpha(1)beta(2)gamma(2L) or for alpha(1)beta(2) recombinant GABA(A)R subunits. Patch clamp techniques were used in the whole-cell mode to evaluate the effect of N(2)O and Xe on GABA-induced currents. A piezo-driven "liquid filament switch" was used for fast application. Both N(2)O (100%, 29.2 mM) and Xe (100%, 3.9 mM) reversibly increased GABA-induced currents through the alpha(1)ss(2)gamma(2L) and the alpha(1)beta(2) GABA(A)R channels. The potentiating effect of N(2)O or Xe on peak currents was prominent at small GABA concentrations (10(-7) to 10(-5) M). The addition of N(2)O or Xe increased the efficacy of GABA (10(-7) to 10(-3) M). Both N(2)O and Xe significantly decreased the risetime((10%-90%)) of the currents elicited by small GABA concentrations. At the concentrations used, neither N(2)O nor Xe had an intrinsic effect. We conclude that, similar to other anesthetics, both N(2)O and Xe increase the efficacy of GABA at the GABA(A)R and enhance inhibitory GABAergic synaptic transmission.

Two conserved arginines in the extracellular N-terminal domain of the GABA(A) receptor alpha(5) subunit are crucial for receptor function.

The gamma-aminobutyric acid (GABA) binding pocket within the GABA(A) receptor complex has been suggested to contain arginine residues. The aim of this study was to test this hypothesis by mutating arginine residues potentially contributing to the formation of a GABA binding pocket. Thus, six arginines conserved in human GABA(A) receptor alpha subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two nonconserved arginines (79 and 190), all located in the extracellular N-terminal segment of the alpha(5) subunit, were substituted by lysines. The individual alpha(5) subunit mutants were coexpressed with human beta(2) and gamma(2s) GABA(A) receptor subunits in Chinese hamster ovary cells by transient transfection. Electrophysiological whole-cell patch-clamp recordings show that, of the eight arginine residues tested, the two arginines at positions 70 and 123 appear to be essential for the GABA-gated chloride current because the EC(50) values of the two mutant constructs increase >100-fold compared with the wild-type alpha(5),beta(2), gamma(2s) GABA(A) receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.

Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination.

Some steroids are synthesized within the central and peripheral nervous system, mostly by glial cells. These are known as neurosteroids. In the brain, certain neurosteroids have been shown to act directly on the function of membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3alpha,5alpha-reduced metabolite 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) activates the gamma-aminobutyric acid receptor complex A (GABA-R(A)). Besides these effects, neurosteroids also regulate important glial functions such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.

Anti-seizure effects of progesterone and 3alpha,5alpha-THP in kainic acid and perforant pathway models of epilepsy.

The mechanism by which progesterone has its anti-seizure effects is unknown. Progesterone has a high affinity for intracellular progestin receptors, but has weak actions at gamma-aminobutyric acid (GABA)(A) receptors complexes. The progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) is devoid of activity at intracellular progestin receptors but is a highly effective modulator of GABA(A) receptor complexes. Whether progesterones anti-seizure actions are due to effects of progesterone itself or its metabolite 3alpha,5alpha-THP was investigated. In experiment 1, 25 ovariectomized Long-Evans rats were subcutaneously (s.c.) injected with 0.0, 4.0 or 8.0 mg/kg progesterone or 3alpha,5alpha-THP, 10 min prior to systemic administration of 32 mg/kg kainic acid. Four and 8.0 mg/kg progesterone significantly reduced the duration of partial and full seizures, without influencing the latency to partial or full seizures, or the number of partial or full seizures. 3alpha, 5alpha-THP (4.0 mg/kg) significantly increased the latency to initial partial seizure, and decreased the number and duration of partial seizures. In experiment 2, 60 ovariectomized Long-Evans rats were stereotaxically implanted with bipolar electrodes into the perforant pathway. Prior to perforant pathway stimulation, rats were s.c. injected with either progesterone (4.0 mg/kg, n = 12), 3alpha, 5alpha-THP (4.0 mg/kg, n = 13), progesterone (4.0 mg/kg)+4MA (10.0 mg of a 5alpha-reductase inhibitor, 17b-N, N-diethylcarbamoyl-4-methyl-4-aza,5alpha-androstan-3-one, n = 12), 4MA+vehicle (n = 10), or sesame oil vehicle (n = 13). Administration of progesterone or 3alpha, 5alpha-THP, but not vehicle control, P+4MA, or 4MA, resulted in significant decreases in partial seizures. In experiment 3, whole brain progesterone and 3alpha,5alpha-THP were measured by radioimmunoassay in additional rats (n = 66) administered the hormonal milieu indicated in experiments 1 and 2. Data suggest anti-seizure effects of progesterone may be due, in part, to metabolism to 3alpha,5alpha-THP and subsequent actions at GABA(A) receptor complexes.

A novel strategy for the treatment of cocaine addiction.

Cocaine's addictive liability has been linked to its pharmacologic actions on mesotelencephalic dopamine (DA) reinforcement/reward pathways in the central nervous system (CNS). Dopaminergic transmission within these pathways is modulated by gamma-aminobutyric acid (GABA). With this knowledge, we examined the utility of gamma vinylGABA (GVG), a selective and irreversible inhibitor of GABA-transaminase (GABA-T) known to potentiate GABAergic inhibition, to alter cocaine's biochemical effects as well as its effects on behaviors associated with these biochemical changes. GVG significantly attenuated cocaine-induced increases in neostriatal synaptic DA in the non-human primate (baboon) brain as assessed by positron emission tomography (PET) and abolished both the expression and acquisition of cocaine-induced conditioned place preference (CPP). It had no effect on CPP for a food reward, the delivery of cocaine to the brain or locomotor activity. These findings suggest the possible therapeutic utility in cocaine addiction of a pharmacologic strategy targeted at the GABAergic neurotransmitter system, a system distinct from but functionally linked to the DA mesotelencephalic reward/reinforcement system. However, rather than targeting the GABA receptor complex with a direct GABA agonist, this novel approach with GVG takes advantage of the prolonged effects of an irreversible enzyme inhibitor that raises endogenous GABA levels without the addictive liability associated with GABA agonists acting directly at the receptor itself. Human trials with GVG are currently being developed to directly examine the utility of this novel strategy for the treatment of cocaine addiction.

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor.

1. The gamma-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA(A) receptors are favoured by beta2- or beta3- versus beta1-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (beta2 N290, beta3 N289, beta1 S290). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. 2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at -60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC50 for GABA of 20+/-0.4 microM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (RdlM314N) or a serine (RdlM314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC50 of 4.8+/-0.2 microM. 3. Utilizing the appropriate GABA EC10, etomidate (300 microM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at RdlM314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC50 of 64+/-3 microM and an Emax of 68+/-2% (of the maximum response to GABA). 4. The actions of etomidate at RdlM314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 microM R-(+)-etomidate and S-(-)-etomidate enhancing the current induced by GABA (EC10) to 52+/-6% and 12+/-1% of the GABA maximum respectively. 5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49+/-4% of the GABA maximum) and RdlM314N receptors (53+/-2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5alpha-pregnan-3alpha-ol-20-one (Rdl = 25+/-4% of the GABA maximum) was not altered by this mutation (RdlM314N = 18+/-3% of the GABA maximum). 6. Etomidate acting at beta1 (S290)-containing mammalian GABA(A) receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at RdlM314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to RdlM314N receptors. 7. Etomidate acting at human alpha6beta3gamma2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA(A) receptor complex. Mutation of the human beta3 subunit asparagine to methionine (beta3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10-300 microM) acting at alpha6beta3 N289Mgamma2L receptors. 8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

The anxiolytic effect of gamma-hydroxybutyrate in the elevated plus maze is reversed by the benzodiazepine receptor antagonist, flumazenil.

The effects of gamma-hydroxybutyrate (GHB), a product of gamma-aminobutyric acid (GABA) metabolism which possesses neuromodulatory properties in brain, were investigated in the elevated plus maze in rats. The number of entries and the time spent in the open arms of the maze were increased by GHB (50, 150, 250 mg/kg i.p.). This is classically considered as indicative of an anxiolytic effect of the drug. There was no sedative effect at these doses as measured by the spontaneous locomotor activity in the actimeter or the total number of arm entries. The anxiolytic properties of GHB were reversed by neither the GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5(H)-5-olylidene acetic acid) (300 mg/kg i.p.), nor the opioid receptor antagonist, naloxone (10 mg/kg i.p.). However the anti-anxiety effect of GHB was antagonized by the benzodiazepine receptor antagonist, flumazenil (10 mg/kg i.p.), suggesting an interaction of GHB with the GABA(A) receptor complex which mediates the anti-anxiety effect of benzodiazepines.

Roles of neuroactive amino acids in ammonia neurotoxicity.

Many neurologic disorders are related to congenital or acquired hyperammonemia (HA). Advanced symptoms of HA range from seizures in acute stages to stupor and coma in more chronic conditions, manifesting variable imbalance between the inhibitory and excitatory neurotransmission. Evidence obtained with the use of experimental HA models suggests that acute neurotoxic effects of ammonia are mediated by overactivation of ionotropic glutamate (GLU) receptors, mainly the N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree the KA/AMPA receptors. NMDA receptor-mediated neurotoxicity may be potentiated by impaired control of their function by metabotropic GLU receptors, which are inactivated by ammonia. Prolonged overactivation of the NMDA receptors upon extended ammonia exposure causes their downregulation. The GLU receptor changes may be related to their excessive exposure to extrasynaptic GLU. Ammonia promotes GLU accumulation in the extrasynaptic space by enhancing its release from neurons, and/or by decreasing its reuptake to the nerve endings and astrocytes, where the effect results from inactivation (downregulation) of the astrocytic glutamate transporter GLT1. Excitotoxic effects of ammonia are augmented by increased synthesis of nitric oxide (NO), which is associated with NMDA receptor activation and/or increased synaptic transport of arginine (ARG). A shift toward neural inhibition is promoted by positive modulation of the gamma-aminobutyric acid (GABA)ergic tone resulting from excessive accumulation in the brain of endogenous central benzodiazepine receptor agonists, and from upregulation of astrocytic peripheral benzodiazepine receptors leading to elevated levels of prognenelone-derived neurosteroids, which positively modulate the GABA(A) receptor complex. Inhibitory neurotransmission may also be favored by enhanced release from astrocytes of an inhibitory amino acid, taurine.

Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference.

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Molecular and Pharmacological Characterization of Native Cortical γ-Aminobutyric AcidA Receptors Containing Both α1 and α3 Subunits

We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical alpha1-alpha3 gamma-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of alpha1 and alpha3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-alpha1- or anti-alpha3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the alpha1-alpha3 GABAA receptors represented 54.7 +/- 5.0 and 23.6 +/- 3.3% of the alpha3 and alpha1 populations, respectively. Therefore, alpha1 and alpha3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these alpha1-alpha3 GABAA receptors from the cortex constitute a large proportion of the total alpha3 population and a relatively minor component of the alpha1 population. The pharmacological analysis of the alpha1- or alpha3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both alpha1 and alpha3 subunits, with alpha1 and alpha3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-alpha1-anti-alpha3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of alpha1 and alpha3 subunits, associated with beta2/3, and gamma2 subunits. The pharmacological analysis of alpha1-alpha3 GABAA receptors demonstrated that, despite the fact that each alpha subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the alpha1 subunit is pharmacologically predominant over the alpha3 subunit. These results indicate the existence of active and nonactive alpha subunits in the native alpha1-alpha3 GABAA receptors from rat cortex.

Molecular genetic analysis of the role of GABAergic systems in the behavioral and cellular actions of alcohol.

Recent studies implicate the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in many neurochemical actions of ethanol and a variety of behavioral responses to acute and chronic ethanol treatment. However, the molecular mechanisms responsible for genetic differences in initial neurochemical or behavioral sensitivity to ethanol, and adaptation following chronic or repeated ethanol administration, remain to be elucidated. Pharmacogenetic research will increasingly move toward mapping, cloning, identification, and functional analysis of the genes underlying the actions of ethanol. The approaches discussed here permit molecular analysis of both known and previously unknown genes regulating behavioral sensitivity to ethanol. The synthesis of molecular methods and behavioral genetics offers immediate hope for delineating the role of the GABA(A) receptor complex, and other determinants of GABAergic neurotransmission, in determining genetic variation in behavioral responses to ethanol.

GABA physiology: modulation by benzodiazepines and hormones.

This review compares the ability of acute and chronic benzodiazepine treatments (BZs), gonadal hormone treatments, and neurosteroids to modify gamma-aminobutyric acid (GABA) physiologic responses in animals. Both BZ ligands and certain "neurosteroid" derivatives of steroid hormones can modulate GABA responses through direct interactions with the GABA receptor complex. Fluctuations in gonadal steroids can be anticonvulsant or proconvulsant, anxiolytic or anesthetic, suggesting a pharmacologic profile comparable to that of the BZs. A comparison of neuronal physiological responses in spinal cord, hippocampus, amygdala, hypothalamus-preoptic area, cerebellum, dorsal raphe, locus coeruleus, and cortex indicates that both acute and chronic BZ treatments produce region-specific effects on in vivo GABAergic responses. This appears to be based on regional variability in intrinsic sensitivity to GABA and/or BZ ligands, the level of ongoing GABA neurotransmission in a region, and the indirect influences of BZs on afferent inputs to an area. Regionally specific adaptations to chronic BZ treatments that lead to the development of tolerance include the following: intrinsic subsensitivity to GABA, reduced activity in GABAergic circuits (e.g., reduced recurrent inhibition in hippocampus), attenuated responses to BZs without alterations in GABA sensitivity, modulation of the intrinsic properties of neurons, and alterations in the activity of non-GABAergic afferent inputs. The in vivo evidence that neurosteroid derivatives modify physiological responses to GABA is also beginning to emerge. Progesterone, through conversion to its neurosteroid metabolite 3 alpha-OH DHP, potentiates GABA responses of cerebellar Purkinje cells. This neurosteroid also enhances GABA responses in hippocampal slices, including recurrent inhibition, in a BZ-like manner. Despite the evidence that fluctuations in estrogen levels can modulate several aspects of GABA neurotransmission, neither in vivo estrogen treatments nor in vitro administration of estrogen to brain slices clearly modifies physiological GABA responses. Although estrogens alter excitability in several areas, these changes are associated with estrogenic effects on responses to excitatory neurotransmitters or inputs. Relatively few studies have examined the in vivo influences of androgenic steroids on GABA responses.