Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter of the mammalian brain, can induce coma. Outside the central nervous system it is synthesized by gut bacteria and catabolized largely in the liver. GABA and its agonists, as well as benzodiazepines and barbiturates, induce neural inhibition as a consequence of their interaction with specific binding sites for each of these classes of neuroactive substances on the GABA receptor complex of postsynaptic neurons. In a rabbit model of acute liver failure: (i) the pattern of postsynaptic neuronal activity in hepatic coma, as assessed by visual evoked potentials, is identical to that associated with coma induced by drugs which activate the GABA neurotransmitter system (benzodiazepines, barbiturates, and GABA agonists); (ii) the levels of GABA-like activity in peripheral blood plasma increase appreciably before the onset of hepatic encephalopathy, due at least in part to impaired hepatic extraction of gut-derived GABA from portal venous blood; (iii) the blood-brain barrier becomes abnormally permeable to an isomer of GABA, alpha-amino-isobutyric acid, before the onset of hepatic encephalopathy; and (iv) hepatic coma is associated with an increase in the density of receptors for GABA and benzodiazepines in the brain. These findings are the bases of the following hypotheses: (i) when the liver fails, gut-derived GABA in plasma crosses an abnormally permeable blood-brain barrier and by mediating neural inhibition contributes to hepatic encephalopathy; (ii) an increased number of GABA receptors in the brain found in liver failure increases the sensitivity of the brain to GABA-ergic neural inhibition; and (iii) an increased number of drug binding sites mediates the increased sensitivity to benzodiazepines and barbiturates observed in liver failure by permitting increased drug effect.
γ-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system (CNS). It exerts its rapid inhibitory action mostly through GABAA receptors, which are targets for benzodiazepines, barbiturates, neuroactive steroids and distinct anticonvulsive agents. There is considerable evidence that dysfunction of GABAA receptors or dysregulation of GABA concentrations in the CNS (or both) plays an important role in the pathophysiology of panic disorder. Currently, benzodiazepines are the only drugs directly targeting the GABAA receptors that are approved for the treatment of anxiety disorders. Because of their well-known anxiolytic effects, they are widely used in this setting, but side effects limit their use in long-term treatment. The question of whether drugs that selectively increase GABA concentrations in the CNS could improve symptoms of anxiety has been discussed. Recent investigations by our group have demonstrated that enhancement of endogenous GABA (through blockade of GABA transaminase by vigabatrin or through inhibition of GABA transporters by tiagabine) exerts anxiolytic effects on experimentally induced panic. Our studies in healthy volunteers have shown that both compounds lead to a significant reduction in panic symptoms elicited by cholecystokinin-tetrapeptide. Moreover, benzodiazepine-like effects on the activity of the hypothalamic–pituitary–adrenal axis have been observed in association with vigabatrin treatment. Small open studies in patients with panic disorder also showed an improvement in panic and anxiety with both compounds. This review summarizes our recent research on the effects of selective GABAergic treatment in experimentally induced panic and outlines the possible role of compounds targeting the GABA binding site of the GABAA–benzodiazepine receptor for the treatment of panic and anxiety.
gamma-aminobutyric acid; panic; anxiety; vigabatrin; tiagabine; alprazolam
γ-aminobutyric acid (GABA) plays important roles in the central nervous system, acting as a neurotransmitter on both ionotropic ligand-gated Cl--channels, and metabotropic G-protein coupled receptors (GPCRs). These two types of receptors called GABAA (and C) and GABAB are the targets of major therapeutic drugs such as the anxiolytic benzodiazepines, and antispastic drug baclofen (lioresal®), respectively. Although the multiplicity of GABAA receptors offer a number of possibilities to discover new and more selective drugs, the molecular characterization of the GABAB receptor revealed a unique, though complex, heterodimeric GPCR. High throughput screening strategies carried out in pharmaceutical industries, helped identifying new compounds positively modulating the activity of the GABAB receptor. These molecules, almost devoid of apparent activity when applied alone, greatly enhance both the potency and efficacy of GABAB agonists. As such, in contrast to baclofen that constantly activates the receptor everywhere in the brain, these positive allosteric modulators induce a large increase in GABAB-mediated responses only WHERE and WHEN physiologically needed. Such compounds are then well adapted to help GABA to activate its GABAB receptors, like benzodiazepines favor GABAA receptor activation. In this review, the way of action of these molecules will be presented in light of our actual knowledge of the activation mechanism of the GABAB receptor. We will then show that, as expected, these molecules have more pronounced in vivo responses and less side effects than pure agonists, offering new potential therapeutic applications for this new class of GABAB ligands.
Baclofen; anxiety; drug addiction; allosteric modulators; class C GPCRs.
OBJECTIVE: Hepatic encephalopathy (HE) is a complex neuropsychiatric disorder secondary to acute or chronic liver failure. Although the exact causes of HE have not been clarified, enhanced central nervous system inhibition at the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex, mediated by increased levels of endogenous benzodiazepine-receptor ligands (BZRL), has been proposed. Research exploring this hypothesis has yielded contradictory findings. This study evaluated the presence and levels of BZRL in plasma from patients with HE and 3 comparison groups. DESIGN: Cross-sectional study. PATIENTS: Twenty-four patients with HE, 10 patients with liver cirrhosis without encephalopathy (LC), 4 patients with uremic encephalopathy (UE), and 9 healthy subjects. INTERVENTIONS: Radio-receptor assay of plasma samples from patients and controls. MAIN OUTCOME MEASURES: Plasma levels of BZRL. RESULTS: The patients in the HE group had significantly higher plasma BZRL levels than the patients with UE and the healthy subjects, but not than those with LC, in whom these compounds were also detected in significant concentrations. When patients were classified according to the severity of HE, plasma of BZRL showed a modest correlation with stage of severity (r = 0.37). Interestingly, approximately one-third of the patients with HE did not have detectable levels of BZRL. CONCLUSION: Endogenous BZRL may play a role in the pathogenesis of HE, although neuropsychiatric symptoms in HE are difficult to explain in terms of these compounds alone.
Sleep is a crucial biological process is regulated through complex interactions between multiple brain regions and neuromodulators. As sleep disorders can have deleterious impacts on health and quality of life, a wide variety of pharmacotherapies have been developed to treat conditions of excessive wakefulness and excessive sleepiness. The neurotransmitter norepinephrine (NE), through its involvement in the ascending arousal system, impacts the efficacy of many wake- and sleep-promoting medications. Wake-promoting drugs such as amphetamine and modafinil increase extracellular levels of NE, enhancing transmission along the wake-promoting pathway. GABAergic sleep-promoting medications like benzodiazepines and benzodiazepine-like drugs that act more specifically on benzodiazepine receptors increase the activity of GABA, which inhibits NE and the wake-promoting pathway. Melatonin and related compounds increase sleep by suppressing the activity of the neurons in the brain’s circadian clock, and NE influences the synthesis of melatonin. Antihistamines block the wake-promoting effects of histamine, which shares reciprocal signaling with NE. Many antidepressants that affect the signaling of NE are also used for treatment of insomnia. Finally, adrenergic antagonists that are used to treat cardiovascular disorders have considerable sedative effects. Therefore, NE, long known for its role in maintaining general arousal, is also a crucial player in sleep pharmacology. The purpose of this review is to consider the role of NE in the actions of wake- and sleep-promoting drugs within the framework of the brain arousal systems.
norepinephrine; locus coeruleus; sleep; wake; arousal
An imbalance between inhibitory and excitatory neurotransmission has been
proposed to contribute to altered brain function in individuals with Down
syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory
neurotransmitter in the central nervous system and accordingly treatment with
GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a
genetic model for DS. However, GABA-A antagonists are also convulsant which
preclude their use for therapeutic intervention in DS individuals. Here, we have
evaluated safer strategies to release GABAergic inhibition using a
GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype
(α5IA). We demonstrate that α5IA restores learning and memory functions of
Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks.
Furthermore, we show that following behavioural stimulation, α5IA enhances
learning-evoked immediate early gene products in specific brain regions involved
in cognition. Importantly, acute and chronic treatments with α5IA do not induce
any convulsant or anxiogenic effects that are associated with GABA-A antagonists
or non-selective inverse agonists of the GABA-A-benzodiazepine receptors.
Finally, chronic treatment with α5IA did not induce histological alterations in
the brain, liver and kidney of mice. Our results suggest that non-convulsant
α5-selective GABA-A inverse agonists could improve learning and memory deficits
in DS individuals.
Down syndrome; GABA-A; inverse agonist; learning; memory; therapy
Zolpidem is a non-benzodiazepine sedative/hypnotic that acts at GABAA receptors to influence inhibitory neurotransmission throughout the central nervous system. A great deal is known about the behavioral effects of this drug in humans and laboratory animals, but little is known about zolpidem’s specific effects on neurochemistry in vivo.
We evaluated how acute administration of zolpidem affected levels of GABA, glutamate, glutamine, and other brain metabolites.
Proton magnetic resonance spectroscopy (1H MRS) at 4 Tesla was employed to measure the effects of zolpidem on brain chemistry in 19 healthy volunteers. Participants underwent scanning following acute oral administration of a therapeutic dose of zolpidem (10 mg) in a within-subject, single-blind, placebo-controlled, single-visit study. In addition to neurochemical measurements from single voxels within the anterior cingulate (ACC) and thalamus, a series of questionnaires were administered periodically throughout the experimental session to assess subjective mood states.
Zolpidem reduced GABA levels in the thalamus, but not the ACC. There were no treatment effects with respect to other metabolite levels. Self-reported ratings of “dizzy”, “nauseous”, “confused”, and “bad effects” were increased relative to placebo, as were ratings on the sedation/intoxication (PCAG) and psychotomimetic/dysphoria (LSD) scales of the Addiction Research Center Inventory. Moreover, there was a significant correlation between the decrease in GABA and “dizzy”.
Zolpidem engendered primarily dysphoric-like effects and the correlation between reduced thalamic GABA and “dizzy” may be a function of zolpidem’s interaction with α1GABAA receptors in the cerebellum, projecting through the vestibular system to the thalamus.
zolpidem; spectroscopy; GABA; glutamate; glutamine; thalamus; anterior cingulate
GABAA receptors mediate the action of many clinically important drugs interacting with different binding sites. For some potential binding sites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular α1+β3− interface, which is homologous to the benzodiazepine binding site at the α1+γ2− interface. On screening of >100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3–c] quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents atα1β3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at α1β3γ2 and α1β3 receptors. By mutating pocket-forming amino acid residues at the α1+ and the β3− side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethio-sulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a generalin activation of GABAA receptors, because the GABA-enhancing effect of ROD 188 or the steroid α-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the α and β subunit types forming the interface. CGS 9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the α+β− interface of GABAA receptors. Since such binding sites are present at αβ, αβγ, and αβδ receptors, such drugs will have a much broader action than benzodiazepines and might become clinical important for the treatment of epilepsy.
We review recent findings pertaining to several environmental agents (ethanol, phencyclidine, ketamine, nitrous oxide, barbiturates, benzodiazepines, halothane, isoflurane, and propofol) that have the potential to delete large numbers of neurons from the developing brain by a newly discovered mechanism involving interference in the action of neurotransmitters [glutamate and gamma-amino butyric acid (GABA) at (italic)N(/italic)-methyl-d-aspartate (NMDA)] and GABA(subscript)A(/subscript) receptors during the synaptogenesis period, also known as the brain growth-spurt period. Transient interference (lasting >= 4 hr) in the activity of these transmitters during the synaptogenesis period (the last trimester of pregnancy and the first several years after birth in humans) causes millions of developing neurons to commit suicide (die by apoptosis). Many of these agents are drugs of abuse (ethanol is a prime example) to which the human fetal brain may be exposed during the third trimester by drug-abusing mothers. Ethanol triggers massive apoptotic neurodegeneration in the developing brain by interfering with both the NMDA and GABA(subscript)A(/subscript) receptor systems, and this can explain the reduced brain mass and lifelong neurobehavioral disturbances associated with intrauterine exposure of the human fetus to ethanol (fetal alcohol syndrome). Exposure of the immature brain in a medical treatment context is also of concern because many of these agents are drugs used frequently as sedatives, tranquilizers, anticonvulsants, or anesthetics in pediatric and/or obstetrical medicine. Because this is a newly discovered mechanism, further research will be required to fully ascertain the nature and degree of risk posed by exposure of the developing human brain to environmental agents that act by this mechanism.
Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design.
γ-Amino butyric acid (GABA) is a well-characterized inhibitory neurotransmitter in the central nervous system, which may also stimulate nonvesicular release of other neurotransmitters under certain conditions. We have recently reported that γ-vinyl GABA (GVG), an irreversible GABA transaminase inhibitor, elevates extracellular GABA but fails to alter dopamine release in the nucleus accumbens (NAc).
Here, we investigated the mechanism(s) by which GVG elevates extracellular GABA levels and whether GVG also alters glutamate release in the NAc.
Materials and methods
In vivo microdialysis was used to simultaneously measure extracellular NAc GABA and glutamate before and after GVG administration in freely moving rats.
Systemic administration of GVG or intra-NAc local perfusion of GVG significantly increased extracellular NAc GABA and glutamate. GVG-enhanced GABA was completely blocked by intra-NAc local perfusion of 5-nitro-2, 3-(phenylpropylamino)-benzoic acid (NPPB), a selective anion channel blocker and partially blocked by SKF89976A, a type 1 GABA transporter inhibitor. GVG-enhanced glutamate was completely blocked by NPPB or SKF89976A. Tetrodotoxin, a voltage-dependent Na+-channel blocker, failed to alter GVG-enhanced GABA and glutamate.
These data suggest that GVG-enhanced extracellular GABA and glutamate are mediated predominantly by the opening of anion channels and partially by the reversal of GABA transporters. Enhanced extracellular glutamate may functionally attenuate the pharmacological action of GABA and prevent enhanced GABA-induced excess inhibition.
γ-Vinyl GABA; GABA; Glutamate; Anion channel; GABA transporter; Nucleus accumbens
Evidence indicates that synchronization of cortical activity at gamma-band frequencies, mediated through GABA-A receptors, is important for perceptual/cognitive processes. To study GABA signaling in vivo, we recently used a novel positron emission tomography (PET) paradigm measuring the change in binding of the benzodiazepine (BDZ) site radiotracer [11C]flumazenil associated with increases in extracellular GABA induced via GABA membrane transporter (GAT1) blockade with tiagabine. GAT1 blockade resulted in significant increases in [11C]flumazenil binding potential (BPND) over baseline in the major functional domains of the cortex, consistent with preclinical studies showing that increased GABA levels enhance the affinity of GABA-A receptors for BDZ ligands. In the current study we sought to replicate our previous results and to further validate this approach by demonstrating that the magnitude of increase in [11C]flumazenil binding observed with PET is directly correlated with tiagabine dose. [11C]flumazenil distribution volume (VT) was measured in 18 healthy volunteers before and after GAT1 blockade with tiagabine. Two dose groups were studied (n = 9 per group; Group I: tiagabine 0.15 mg/kg; Group II: tiagabine 0.25 mg/kg). GAT1 blockade resulted in increases in mean (± SD) [11C]flumazenil VT in Group II in association cortices (6.8±0.8 mL g−1 vs. 7.3±0.4 mL g−1;p = 0.03), sensory cortices (6.7±0.8 mL g−1 vs. 7.3±0.5 mL g−1;p = 0.02) and limbic regions (5.2±0.6 mL g−1 vs. 5.7±0.3 mL g−1;p = 0.03). No change was observed at the low dose (Group I). Increased orbital frontal cortex binding of [11C]flumazenil in Group II correlated with the ability to entrain cortical networks (r = 0.67, p = 0.05) measured via EEG during a cognitive control task. These data provide a replication of our previous study demonstrating the ability to measure in vivo, with PET, acute shifts in extracellular GABA.
Accumulating evidence indicates that synchronization of cortical neuronal activity at γ-band frequencies is important for various types of perceptual and cognitive processes and that GABA-A receptor-mediated transmission is required for the induction of these network oscillations. In turn, the abnormalities in GABA transmission postulated to play a role in psychiatric conditions such as schizophrenia might contribute to the cognitive deficits seen in this illness. We measured the ability to increase GABA in eight healthy subjects by comparing the binding of [11C]flumazenil, a positron emission tomography (PET) radiotracer specific for the benzodiazepine (BDZ) site, at baseline and in the presence of an acute elevation in GABA levels through the blockade of the GABA membrane transporter (GAT1). Preclinical work suggests that increased GABA levels enhance the affinity of GABA-A receptors for BDZ ligands (termed ‘GABA shift’). Theoretically, such an increase in the affinity of GABA-A receptors should be detected as an increase in the binding of a GABA-A BDZ-receptor site-specific PET radioligand. GAT1 blockade resulted in significant increases in mean (± SD) [11C]flumazenil-binding potential (BPND) over baseline in brain regions representing the major functional domains of the cerebral cortex: association cortex + 15.2 ± 20.2% (p = 0.05), sensory cortex + 13.5 ± 15.5% (p = 0.03) and limbic (medial temporal lobe, MTL) + 16.4 ± 20.2% (p = 0.03). The increase in [11C]flumazenil-BPND was not accounted for by differences in the plasma-free fraction (fP; paired t-test p = 0.24) or changes in the nonspecific binding (pons VT, p = 0.73). Moreover, the ability to increase GABA strongly predicted (r = 0.85, p = 0.015) the ability to entrain cortical networks, measured through EEG γ synchrony during a cognitive control task in these same subjects. Although additional studies are necessary to further validate this technique, these data provide preliminary evidence of the ability to measure in vivo, with PET, acute fluctuations in extracellular GABA levels and provide the first in vivo documentation of a relationship between GABA neurotransmission and EEG γ-band power in humans.
GABA; PET; [11C]flumazenil; GABA shift; γ-band; oscillations
GABAA receptors are located on the majority of neurons in the central and peripheral nervous system, where they mediate important actions of the neurotransmitter gamma-aminobutyric acid. Early in development the trophic properties of GABA allow a healthy development of the nervous system. Most neurons have a high intracellular Cl-concentration early in life due to the late functional expression of the Cl-pump KCC2, therefore GABA has excitatory effects at this stage. Upon higher expression and activation of KCC2 GABA takes on its inhibitory effects while glutamate functions as the major excitatory neurotransmitter. Like all multisubunit membrane proteins the GABAA receptor is assembled in the ER and travels through the Golgi and remaining secretory pathway to the cell surface, where it mediates GABA actions either directly at the synapses or at extrasynaptic sites responding to ambient GABA to provide a basal tonic inhibitory state. In order to adapt to changing needs and information states, the GABAergic system is highly dynamic. That includes subtype specific trafficking to different locations in the cell, regulation of mobility by interaction with scaffold molecules, posttranslational modifications, that either directly affect channel function or the interaction with other proteins and finally the dynamic exchange between surface and intracellular receptor pools, that either prepare receptors for recycling to the surface or degradation. Here we give an overview of the current understanding of GABAA receptor functional and molecular dynamics that play a major part in maintaining the balance between excitation and inhibition and in changes in network activity.
GABAA receptor; receptor trafficking; receptor clustering; inhibition
Fenamate NSAIDs have several central effects, including anti-epileptic
and neuroprotective actions. The underlying mechanism(s) of these actions are not presently
understood. In this study, the effects of five members of the fenamate NSAID group were
investigated on native ligand-gated ion channels expressed in cultured rat hippocampal neurons.
All fenamates tested, (1–100μM) dose-dependently potentiated GABA-evoked
currents; mefenamic acid (MFA) was the most potent and efficacious and was found to shift the
GABA dose response curve to the left without effect on the maximum amplitude or the GABA Hill
Slope. The modulation of GABA receptors by MFA was not reduced in the presence of the
benzodiazepine antagonist, flumazenil (10μM) and was moderately voltage-dependent. MFA
at concentrations ≥10μM evoked dose-dependent currents in the absence of GABA. These currents were potentiated by diazepam (1μM) and blocked by bicuculline (10μM). The MFA (50μM) current-voltage relationship and reversal potential were similar to that evoked by GABA. MFA (1–100μM) had no effects on sub-maximal glycine, glutamate or NMDA evoked currents. These data show that fenamate NSAIDs are a highly effective class of GABAA receptor modulator and activators.
Anticonvulsant; NSAID; electrophysiology; glutamate receptors; glycine receptors
Flumazenil, an imidazobenzodiazepine, is the first benzodiazepine antagonist and is being used to reverse the adverse pharmacological effects of benzodiazepine. There have been a few reports on the central nevous system side effects with its use. We report a patient with generalized ballism following administration of flumazenil. The mechanism through which flumazenil induced this symptom is unknown. It is conceivable that flumazenil may antagonize the GABA-benzodiazepine receptor complex and induce dopamine hypersensitivity, thus induce dyskinesic symptoms.
GABA, best known as a neurotransmitter in the central nervous system, is also present in various peripheral tissues including the gastrointestinal tract, where there is strong evidence that GABA acts as a transmitter in some intrinsic myenteric neurones. Several studies indicate that the gastric mucosa is one of the sites of action of GABA in the gut. Highly specific anti-GABA antibodies have been used to detect endogenous GABA in the mucosa of the rat gastrointestinal tract, and 3H-GABA uptake followed by autoradiography has been used to localise cells with uptake sites for exogenous GABA. It was found that although GABA immunoreactive nerve fibres are essentially absent from this site, some mucosal cells are strongly GABA-immunoreactive. These cells are common in the pyloric stomach and upper part of the small intestine. The autoradiographic experiments provide evidence that these cells also possess high-affinity GABA uptake sites. These observations raise the possibility that in the gastrointestinal tract GABA acts as a gut hormone in a subpopulation of mucosal endocrine cells, in addition to its role as an enteric neurotransmitter.
The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use γ-aminobutyric acid (GABA) as a neurotransmitter, the principal inhibitory neurotransmitter in the adult central nervous system. Acting via the ionotropic GABAA receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl− influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (for chloride entry) and KCC1-4 (for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cationchloride cotransporters. Previously we reported that the K+/Cl− cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K+/Cl− cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl− levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.
circadian rhythms; GABA; KCC2; KCC3; KCC4; NKCC1; WNK3
The amino acids glutamate and gamma-aminobutyric acid (GABA) have primarily been characterized as the most prevalent excitatory and inhibitory, respectively, neurotransmitters in the vertebrate central nervous system. However, the role of these signaling molecules extends far beyond the synapse. GABA, glutamate, and their complement of receptors are essential signaling molecules that regulate developmental processes in both embryonic and young adult mammals. In this review, we describe the current knowledge on the role of GABA and glutamate in development, focusing on the perinatal cerebellum. We will then present novel data suggesting that GABA depolarizes granule cell precursors via GABAA receptors, which leads to calcium increases in these cells. Finally, we will consider the role of GABA and glutamate signaling on cell proliferation and perhaps neural cancers. From our review of the literature and these data, we hypothesize that GABAA receptors and metabotropic glutamate receptors may be a novel target for the pharmacological regulation of the cerebellar tumors, medulloblastomas.
GABA; GABA receptor; cerebellum; external granule cell layer; migration; glutamate; medulloblastoma
Accumulation of the GABA inhibitory neurotransmitter for rapid delivery into synapses can be accomplished by GAD65 dependent and independent membrane-targeting of GAD67.
The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.
GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABAA receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABAA receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1 hr GABA exposure, followed by a 5 hr incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABAA receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of an secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist.
GABAA receptor; γ-aminobutyric acid; ligand chaperone; endoplasmic reticulum; secretory pathway; GABA transporter; glutamic acid decarboxylase
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 GABA system may be affected in autism. The benzodiazepine binding site on the GABAA receptor complex is an important target for pharmacotherapy and has important clinical implications. The present multiple-concentration ligand-binding study utilized 3H-muscimol and 3H-flunitrazepam to determine the number (Bmax), binding affinity (Kd), and distribution of GABAA 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 GABAA 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. In addition, a trend for a decrease in for the density of benzodiazepine sites was found in the infragranular layers (17.1%) in the autism group. These findings suggest that in the autistic group this downregulation of both benzodiazepine sites and GABAA 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.
Autistic; Anterior Cingulate Cortex; GABA; Post-mortem; Ligand binding
Benzodiazepines have a hypnotic/sedative effect through the inhibitory action of γ-aminobutyric acid type A receptor. Flumazenil antagonizes these effects via competitive inhibition, so it has been used to reverse the effect of benzodiazepines. Recently, flumazenil has been reported to expedite recovery from propofol/remifentanil and sevoflurane/remifentanil anesthesia without benzodiazepines. Endogenous benzodiazepine ligands (endozepines) were isolated in several tissues of individuals who had not received benzodiazepines.
Forty-five healthy unpremedicated patients were randomly allocated to either flumazenil or a control groups. Each patient received either a single dose of 0.3 mg of flumazenil (n = 24) or placebo (n = 21). After drug administration, various recovery parameters and bispectral index (BIS) values in the flumazenil and control groups were compared.
Mean time to spontaneous respiration, eye opening on verbal command, hand squeezing on verbal command, extubation and time to date of birth recollection were significantly shorter in the flumazenil group than in the control group (P = 0.004, 0.007, 0.005, 0.042, and 0.016, respectively). The BIS value was significantly higher in flumazenil group than in the control group beginning 6 min after flumazenil administration.
Administration of a single dose of 0.3 mg of flumazenil to healthy, unpremedicated patients at the end of sevoflurane/fentanyl anesthesia without benzodiazepines resulted in earlier emergence from anesthesia and an increase in the BIS value. This may indicate that flumazenil could have an antagonistic effect on sevoflurane or an analeptic effect through endozepine-dependent mechanisms.
Bispectral index; Endozepine; Fentanyl; Flumazenil; Sevoflurane
γ−Amino butyric acid (GABA) is a primary inhibitory neurotransmitter in the central nervous system, and is classically released by fusion of synaptic vesicles with the plasma membrane or by egress via GABA transporters (GATs). Recently, a GABAergic system comprised of GABAA and GABAB receptors has been identified on airway epithelial and smooth muscle cells that regulate mucus secretion and contractile tone of airway smooth muscle (ASM). In addition, the enzyme that synthesizes GABA, glutamic acid decarboxylase, has been identified in airway epithelial cells; however, the mechanism(s) by which this synthesized GABA is released from epithelial intracellular stores is unknown. We questioned whether any of the four known isoforms of GATs are functionally expressed in ASM or epithelial cells. We detected mRNA and protein expression of GAT2 and -4, and isoforms of glutamic acid decarboxylase in native and cultured human ASM and epithelial cells. In contrast, mRNA encoding vesicular GAT (VGAT), the neuronal GABA transporter, was not detected. Functional inhibition of 3H-GABA uptake was demonstrated using GAT2 and GAT4/betaine–GABA transporter 1 (BGT1) inhibitors in both human ASM and epithelial cells. These results demonstrate that two isoforms of GATs, but not VGAT, are expressed in both airway epithelial and smooth muscle cells. They also provide a mechanism by which locally synthesized GABA can be released from these cells into the airway to activate GABAA channels and GABAB receptors, with subsequent autocrine and/or paracrine signaling effects on airway epithelium and ASM.
vesicular γ–amino butyric acid transporter; 3H–γ–amino butyric acid uptake; immunoblot; RT-PCR
Anxiety and stress-related disorders are among the most common psychiatric disorders. The hippocampus is a crucial brain area involved in the neural circuits of the pathophysiology of anxiety and stress-related disorders, and GABA is one of most important neurotransmitters related to these disorders. An anxiogenic drug and a pharmacological stressor, FG7142 (N-methyl-ß-carboline-3-carboxamide), produces anxiety in humans and experimental animals, acting at the benzodiazepine sites of the GABAA receptors as a partial inverse agonist. This drug as well as immobilization stress produced an increased mRNA in a number of genes, e.g., Btg2 and Adamsts1, in the cortex of rodents. The present study was carried out to clarify the effect of the anxiogenic drug on the gene expressions in the hippocampus and to obtain a new insight into the GABAergic system involved in the pathophysiology of the disorders.
We examined the effects of FG7142 on the gene expression of Btg2 and Adamts1 in the hippocampus of mice using a quantitative RT-PCR method as well as an in situ hybridization method.
The intraperitoneal administration of FG7142 at a dose of 20 mg/kg, but not 10 mg/kg, induced a statistically significant increase in the hippocampal mRNA of both genes in adult mice (postnatal days 56), being blocked by co-administrations of flumazenil (twice of 10 mg/kg, i.p.), an antagonist at the benzodiazepine binding site, while FG7142 failed to produce any change in the gene expressions in infant mice (postnatal days 8). In addition, the in situ hybridization experiment demonstrated an upregulation of the gene expressions restricted to the dentate gyrus of the hippocampus in adult mice.
The present study suggests a functional coupling between the GABAergic system and the transcriptional regulation of the two genes (Btg2 and Adamsts1) in the hippocampus of adult mice, which may play a role in the brain function related to anxiety and stress such as memory of fear.
Anxiety; Hippocampus; FG7142; Flumazenil; Btg2; Adamts1; RT-PCR; GABAA receptors; Dentate gyrus; Development