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1.  The GABA hypothesis of the pathogenesis of hepatic encephalopathy: current status. 
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.
PMCID: PMC2589853  PMID: 6093394
2.  Allosteric Modulators of GABAB Receptors: Mechanism of Action and Therapeutic Perspective 
Current Neuropharmacology  2007;5(3):195-201.
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.
PMCID: PMC2656813  PMID: 19305802
Baclofen; anxiety; drug addiction; allosteric modulators; class C GPCRs.
3.  Selective GABAergic treatment for panic? Investigations in experimental panic induction and panic disorder 
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.
PMCID: PMC1089777  PMID: 15944741
gamma-aminobutyric acid; panic; anxiety; vigabatrin; tiagabine; alprazolam
4.  Zolpidem, a clinical hypnotic that affects electronic transfer, alters synaptic activity through potential GABA receptors in the nervous system without significant free radical generation 
Zolpidem (trade name Ambien) has attracted much interest as a sleep-inducing agent and also in research. Attention has been centered mainly on receptor binding and electrochemistry in the central nervous system which are briefly addressed herein. A novel integrated approach to mode of action is presented. The pathways to be discussed involve basicity, reduction potential, electrostatics, cell signaling, GABA receptor binding, electron transfer (ET), pharmacodynamics, structure activity relationships (SAR) and side effects. The highly conjugated pyridinium salt formed by protonation of the amidine moiety is proposed to be the active form acting as an ET agent. Extrapolation of reduction potentials for related compounds supports the premise that zolpidem may act as an ET species in vivo. From recent literature reports, electrostatics is believed to play a significant role in drug action.
The pyridinium cation displays molecular electrostatic potential which may well play a role energetically or as a bridging mechanism. An SAR analysis points to analogy with other physiologically active xenobiotics, namely benzodiazepines and paraquat in the conjugated iminium category. Inactivity of metabolites indicates that the parent is the active form of zolpidem. Absence of reactive oxygen species and oxidative stress is in line with minor side effects. In contrast, generally, the prior literature contains essentially no discussion of these fundamental biochemical relationships. Pharmacodynamics may play an important role. Concerning behavior at the blood-brain barrier, useful insight can be gained from investigations of the related cationic anesthetics that are structurally related to acetyl choline. Evidently, the neutral form of the drug penetrates the neuronal membrane, with the salt form operating at the receptor. The pathways of zolpidem have several clinical implications since the agent affects sedation, electroencephalographic activity, oxidative metabolites and receptors in the central nervous system. The drug acts at the GABA(A) receptor benzodiazepine site, displaying high and intermediate affinities to various receptor regions. Structural features for tight binding were determined. The sedative and anticonvulsant activities are due to its action on the alpha-1-GABA(A) receptors. One of the common adverse responses to zolpidem is hallucinations. Proposed mechanisms comprise changes in the GABA(A) receptor, pharmacodynamic interactions involving serotonin and neuronal-weak photon emission processes entailing redox phenomena. Reports cite cases of abuse with cravings based on anxiolytic and stimulating actions. It is important to recognize that insight concerning processes at the fundamental, molecular level can translate into beneficial results involving both positive and adverse side effects. In order for this to occur, interdisciplinary interaction is necessary. Suggestions are made for future research aimed at testing the various hypotheses.
PMCID: PMC2763231  PMID: 20046645
zolpidem; electrochemistry; receptors; cell signaling; SAR; neuroscience
5.  Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands 
Nature chemical biology  2012;8(5):455-464.
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.
PMCID: PMC3368153  PMID: 22446838
6.  The GABAA Receptor α+β− Interface: A Novel Target for Subtype Selective Drugs 
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.
PMCID: PMC3182524  PMID: 21248110
7.  Good Night and Good Luck: Norepinephrine in Sleep Pharmacology 
Biochemical pharmacology  2009;79(6):801-809.
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.
PMCID: PMC2812689  PMID: 19833104
norepinephrine; locus coeruleus; sleep; wake; arousal
8.  Interactions between modulators of the GABAA receptor: Stiripentol and benzodiazepines 
European journal of pharmacology  2011;654(2):160-165.
Many patients with refractory epilepsy are treated with polytherapy, and nearly 15% of epilepsy patients receive two or more anti-convulsant agents. The anti-convulsant stiripentol is used as an add-on treatment for the childhood epilepsy syndrome known as severe myoclonic epilepsy in infancy (Dravet Syndrome). Stiripentol has multiple mechanisms of action, both enhancing GABAA receptors and reducing activity of metabolic enzymes that break down other drugs. Stiripentol is typically co-administered with other anti-convulsants such as benzodiazepines which also act through GABAA receptor modulation. Stiripentol slows the metabolism of some of these drugs through inhibition of a variety of cytochrome P450 enzymes, but could also influence their effects on GABAergic neurotransmission. Is it rational to co-administer drugs which can act through the same target? To examine the potential interaction between these modulators, we transiently transfected HEK-293T cells to produce α3β3γ2L or α3β3δ recombinant GABAA receptors. Using whole-cell patch clamp recordings, we measured the response to each benzodiazepine alone and in combination with a maximally effective concentration of stiripentol. We compared the responses to four different benzodiazepines: diazepam, clonazepam, clobazam and norclobazam. In all cases we found that these modulators were equally effective in the presence and absence of stiripentol. The δ-containing receptors were insensitive to modulation by the benzodiazepines, which did not affect potentiation by stiripentol. These data suggest that stiripentol and the benzodiazepines act independently at GABAA receptors and that polytherapy could be expected to increase the maximum effect beyond either drug alone, even without consideration of changes in metabolism.
PMCID: PMC3656601  PMID: 21237147
anti-convulsant; electrophysiology; diazepam; clobazam; norclobazam; clonazepam;  recombinant; patch-clamp; Dravet Syndrome
9.  Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice 
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.
PMCID: PMC3160204  PMID: 21693554
Down syndrome; GABA-A; inverse agonist; learning; memory; therapy
10.  A therapeutic dose of zolpidem reduces thalamic GABA in healthy volunteers: A proton MRS study at 4 Tesla 
Psychopharmacology  2009;203(4):819.
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.
PMCID: PMC2818041  PMID: 19125238
zolpidem; spectroscopy; GABA; glutamate; glutamine; thalamus; anterior cingulate
11.  Ganaxolone improves behavioral deficits in a mouse model of post-traumatic stress disorder 
Allopregnanolone and its equipotent stereoisomer, pregnanolone (together termed ALLO), are neuroactive steroids that positively and allosterically modulate the action of gamma-amino-butyric acid (GABA) at GABAA receptors. Levels of ALLO are reduced in the cerebrospinal fluid of female premenopausal patients with post-traumatic stress disorder (PTSD), a severe, neuropsychiatric condition that affects millions, yet is without a consistently effective therapy. This suggests that restoring downregulated brain ALLO levels in PTSD may be beneficial. ALLO biosynthesis is also decreased in association with the emergence of PTSD-like behaviors in socially isolated (SI) mice. Similar to PTSD patients, SI mice also exhibit changes in the frontocortical and hippocampal expression of GABAA receptor subunits, resulting in resistance to benzodiazepine-mediated sedation and anxiolysis. ALLO acts at a larger spectrum of GABAA receptor subunits than benzodiazepines, and increasing corticolimbic ALLO levels in SI mice by injecting ALLO or stimulating ALLO biosynthesis with a selective brain steroidogenic stimulant, such as S-norfluoxetine, at doses far below those that block serotonin reuptake, reduces PTSD-like behavior in these mice. This suggests that synthetic analogs of ALLO, such as ganaxolone, may also improve anxiety, aggression, and other PTSD-like behaviors in the SI mouse model. Consistent with this hypothesis, ganaxolone (3.75–30 mg/kg, s.c.) injected 60 min before testing of SI mice, induced a dose-dependent reduction in aggression toward a same-sex intruder and anxiety-like behavior in an elevated plus maze. The EC50 dose of ganaxolone used in these tests also normalized exaggerated contextual fear conditioning and, remarkably, enhanced fear extinction retention in SI mice. At these doses, ganaxolone failed to change locomotion in an open field test. Therefore, unlike benzodiazepines, ganaxolone at non-sedating concentrations appears to improve dysfunctional emotional behavior associated with deficits in ALLO in mice and may provide an alternative treatment for PTSD patients with deficits in the synthesis of ALLO. Selective serotonin reuptake inhibitors (SSRIs) are the only medications currently approved by the FDA for treatment of PTSD, although they are ineffective in a substantial proportion of PTSD patients. Hence, an ALLO analog such as ganaxolone may offer a therapeutic GABAergic alternative to SSRIs for the treatment of PTSD or other disorders in which ALLO biosynthesis may be impaired.
PMCID: PMC4161165  PMID: 25309317
ganaxolone; allopregnanolone; selective brain steroidogenic stimulants; 5α-reductase type I; PTSD; PTSD therapy; anxiety disorders; GABAA receptor
12.  The discriminative stimulus effects of midazolam are resistant to modulation by morphine, amphetamine, dizocilpine and γ-butyrolactone in rhesus monkeys 
Psychopharmacology  2011;217(4):495-504.
Although abuse of benzodiazepines alone is uncommon, it is high in polydrug abusers, including those who abuse primarily opioids or stimulants.
This study investigated whether drugs that are abused (e.g., amphetamine) or have mechanisms of action similar to abused drugs (e.g., morphine) alter the discriminative stimulus effects of the benzodiazepine midazolam.
Three rhesus monkeys discriminated 0.56 mg/kg of midazolam while responding under a fixed ratio 10 schedule of food presentation. Dose-effect curves were determined for midazolam alone and in the presence of morphine (opioid receptor agonist), amphetamine (dopamine receptor indirect agonist), dizocilpine (N-methyl-D-aspartic acid receptor antagonist), or γ-butyrolactone (prodrug of γ-hydroxybutyrate, which acts primarily at GABAB receptors).
Doses of midazolam larger than 0.32 mg/kg produced ≥80% midazolam-lever responding. When administered alone, morphine, amphetamine, dizocilpine and γ-butyrolactone did not produce midazolam-lever responding, although large doses of each drug eliminated responding; when administered in combination with midazolam, they did not alter the discriminative stimulus effects of midazolam up to doses that markedly decreased response rates.
The current study demonstrates a lack of modulation of the discriminative stimulus effects of midazolam by morphine, amphetamine, dizocilpine, and γ-butyrolactone. Other effects of benzodiazepines, such as their reinforcing effects, might be altered by these other drugs, or benzodiazepines might modulate the discriminative stimulus or reinforcing effects of the other drugs, which might contribute to the relatively high incidence of benzodiazepine abuse among polydrug abusers.
PMCID: PMC3195358  PMID: 21503606
drug abuse; polydrug abusers; midazolam; drug discrimination; monkeys
13.  Unexpected Properties of δ-Containing GABAA Receptors in Response to Ligands Interacting with the α+ β− Site 
Neurochemical research  2013;39(6):1057-1067.
GABAA receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ β− interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αβ, αβγ and αβδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and β subunits present within the receptors. Whereas the presence of a γ2 subunit within αβγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αβ receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1β2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ β− site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ β− interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1β3δ receptors was not observed at α4,6β3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1β3δ receptors was not observed with another α+ β− modulator, and that the efficacy of modulation by α+ β− ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand.
PMCID: PMC3921099  PMID: 24072672
GABAA; CGS 9895; Pyrazoloquinolinones; α+ β− Binding site; Positive modulators; Null modulators; δ Subunit; Extrasynaptic receptor
14.  Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening: the Consequences of Adding an N-3 Methylcarbamoyl Group to the Diazepine Ring of 2,3-Benzodiazepine Derivatives† 
Biochemistry  2011;50(33):7284-7293.
2,3-Benzodiazepine derivatives are AMPA receptor inhibitors, and they are potential drugs for treating some neurological diseases caused by excessive activity of AMPA receptors. Using a laser-pulse photolysis and rapid solution flow techniques, we characterized the mechanism of action of a 2,3-benzodiazepine derivative, termed BDZ-f, by measuring its inhibitory effect on the channel-opening and channel-closing rate constants as well as the whole-cell current amplitude of the homomeric GluA2Q AMPA receptor channels. We also investigated whether BDZ-f competes with GYKI 52466 for binding to the same site on GluA2Qflip. GYKI 52466 is the prototypic 2,3-benzodiazepine compound, and BDZ-f is the N-3 methylcarbamoyl derivative. We found that BDZ-f is a noncompetitive inhibitor with a slight preference for the closed-channel state of both the flip and the flop variants of GluA2Q. Similar to other 2,3-benzodiazepine compounds that we have previously characterized, BDZ-f inhibits GluA2Qflip by forming an initial, loose intermediate that is partially conducting; however, this intermediate rapidly isomerizes into a tighter, fully inhibitory receptor-inhibitor complex. BDZ-f binds to the same noncompetitive site as GYKI 52466 does. Together, our results show that the addition of an N-3 methylcarbamoyl group to the diazepine ring with the azomethine feature (i.e., GYKI 52466) is what makes BDZ-f more potent and more selective towards the closed-channel conformation than the original GYKI 52466. Our results have significant implications for the structure-activity relationship of the 2,3-benzodiazepine series.
PMCID: PMC3170730  PMID: 21751782
15.  Effect of flumazenil on recovery from anesthesia and the bispectral index after sevoflurane/fentanyl general anesthesia in unpremedicated patients 
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.
PMCID: PMC3272523  PMID: 22323949
Bispectral index; Endozepine; Fentanyl; Flumazenil; Sevoflurane
16.  Electrical Stimulation Therapies for CNS Disorders and Pain are Mediated by Competition Between Different Neuronal Networks in the Brain 
Medical hypotheses  2008;71(5):668-681.
CNS neuronal networks are known to control normal physiological functions, including locomotion and respiration. Neuronal networks also mediate the pathophysiology of many CNS disorders. Stimulation therapies, including localized brain and vagus nerve stimulation, electroshock, and acupuncture, are proposed to activate “therapeutic” neuronal networks. These therapeutic networks are dormant prior to stimulatory treatments, but when the dormant networks are activated they compete with pathophysiological neuronal networks, disrupting their function. This competition diminishes the disease symptoms, providing effective therapy for otherwise intractable CNS disorders, including epilepsy, Parkinsons disease, chronic pain, and depression. Competition between stimulation-activated therapeutic networks and pathophysiological networks is a major mechanism mediating the therapeutic effects of stimulation. This network interaction is hypothesized to involve competition for “control” of brain regions that contain high proportions of conditional multireceptive (CMR) neurons. CMR regions, including brainstem reticular formation, amygdala, and cerebral cortex, have extensive connections to numerous brain areas, allowing these regions to participate potentially in many networks. The participation of CMR regions in any network is often variable, depending on the conditions affecting the organism, including vigilance states, drug treatment, and learning. This response variability of CMR neurons is due to the high incidence of excitatory postsynaptic potentials that are below threshold for triggering action potentials. These subthreshold responses can be brought to threshold by blocking inhibition or enhancing excitation via the paradigms used in stimulation therapies. Participation of CMR regions in a network is also strongly affected by pharmacological treatments (convulsant or anesthetic drugs) and stimulus parameters (strength and repetition rate). Many studies indicate that treatment of unanesthetized animals with antagonists (bicuculline and strychnine) of inhibitory neurotransmitter (GABA or glycine) receptors can cause CMR neurons to become consistently responsive to external inputs (e.g. peripheral nerve, sensory, or electrical stimuli in the brain) to which these neurons did not previously respond. Conversely, agents that enhance GABA-mediated inhibition (e.g. barbiturates and benzodiazepines) or antagonize glutamate-mediated excitation (e.g. ketamine) can cause CMR neurons to become unresponsive to inputs to which they responded previously. The responses of CMR neurons exhibit extensive short-term and long-term plasticity, which permits them to participate to a variable degree in many networks. Short-term plasticity subserves termination of disease symptoms, while long-term plasticity in CMR regions subserves symptom prevention. This network interaction hypothesis has value for future research in CNS disease mechanisms and also for identifying therapeutic targets in specific brain networks for more selective stimulation and pharmacological therapies.
PMCID: PMC2650838  PMID: 18762389
acupressure; acupuncture; amygdala; anesthesia; anxiety; barbiturate; benzodiazepine; brain stimulation; CMR; conditional multireceptive neurons; convulsant drugs; depression; electroshock; epilepsy; hypnosis; ketamine; kindling; learning; learning; memory; neuronal networks; pain; placebo; reticular formation; SUDEP; TENS; vagus nerve stimulation
17.  Environmental agents that have the potential to trigger massive apoptotic neurodegeneration in the developing brain. 
Environmental Health Perspectives  2000;108(Suppl 3):383-388.
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.
PMCID: PMC1637813  PMID: 10852832
18.  Chronic Benzodiazepine-induced reduction in GABAA receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection 
Neuroscience  2008;157(1):153-163.
One week oral flurazepam (FZP) administration in rats results in reduced GABAA receptor-mediated synaptic transmission in CA1 pyramidal neurons associated with benzodiazepine tolerance in vivo and in vitro. Since voltage-gated calcium channel (VGCC) current density is enhanced 2-fold during chronic FZP treatment, the role of L-type VGCCs in regulating benzodiazepine-induced changes in CA1 neuron GABAA receptor-mediated function was evaluated. Nimodipine (10 mg/kg, i.p.) or vehicle (0.5 % Tween 80, 2 ml/kg) was injected 1 day after ending FZP treatment and 24 hours prior to hippocampal slice preparation for measurement of mIPSC characteristics and in vitro tolerance to zolpidem. The reduction in GABAA receptor-mediated mIPSC amplitude and estimated unitary channel conductance measured 2 days after drug removal was no longer observed following prior nimodipine injection. However, the single nimodipine injection failed to prevent in vitro tolerance to zolpidem's ability to prolong mIPSC decay in FZP-treated neurons, suggesting multiple mechanisms may be involved in regulating GABAA receptor-mediated synaptic transmission following chronic FZP administration. As reported previously in recombinant receptors, nimodipine inhibited synaptic GABAA receptor currents only at high concentrations (>30 μM), significantly greater than attained in vivo (1 μM) 45 min after a single antagonist injection. Thus, the effects of nimodipine were unlikely to be related to direct effects on GABAA receptors. As with nimodipine injection, buffering intracellular free [Ca2+] with BAPTA similarly prevented the effects on GABAA receptor-mediated synaptic transmission, suggesting intracellular Ca2+ homeostasis is important to maintain GABAA receptor function. The findings further support a role for activation of L-type VGCCs, and perhaps other Ca2+-mediated signaling pathways, in the modulation of GABAA receptor synaptic function following chronic benzodiazepine administration, independent of modulation of the allosteric interactions between benzodiazepine and GABA binding sites.
PMCID: PMC2586180  PMID: 18805463
L-type voltage-gated calcium channels; flurazepam; tolerance; zolpidem; unitary channel conductance; BAPTA
19.  Discriminative stimulus effects of pregnanolone in rhesus monkeys 
Psychopharmacology  2013;231(1):10.1007/s00213-013-3218-2.
Neuroactive steroids and benzodiazepines can positively modulate GABA by acting at distinct binding sites on synaptic GABAA receptors. Although these receptors are thought to mediate the behavioral effects of both benzodiazepines and neuroactive steroids, other receptors (e.g., extrasynaptic GABAA, NMDA, σ1, or 5-HT3 receptors) might contribute to the effects of neuroactive steroids, resulting in differences among positive modulators.
The current study established the neuroactive steroid pregnanolone as a discriminative stimulus to determine whether actions in addition to positive modulation of synaptic GABAA receptors might contribute to its discriminative stimulus effects.
Four rhesus monkeys discriminated 5.6 mg/kg pregnanolone while responding under a fixed-ratio 10 schedule of stimulus-shock termination.
Positive modulators acting at benzodiazepine, barbiturate, or neuroactive steroid sites produced ≥80% pregnanolone-lever responding, whereas drugs acting primarily at receptors other than synaptic GABAA receptors, such as extrasynaptic GABAA, NMDA, σ1, and 5-HT3 receptors, produced vehicle-lever responding. Flumazenil antagonized the benzodiazepines midazolam and flunitrazepam, with Schild analyses yielding slopes that did not deviate from unity and pA2 values of 7.39 and 7.32, respectively. Flumazenil did not alter the discriminative stimulus effects of pregnanolone.
While these results do not exclude the possibility that pregnanolone acts at receptors other than synaptic GABAA receptors, they indicate a primary if not exclusive role of synaptic GABAA receptors in its discriminative stimulus effects. Reported differences in the chronic effects of benzodiazepines and neuroactive steroids are not due to differences in their actions at synaptic GABAA receptors.
PMCID: PMC3882199  PMID: 23949204
pregnanolone; benzodiazepines; drug discrimination; rhesus monkeys
20.  GABA Acts as a Ligand Chaperone in the Early Secretory Pathway to Promote Cell Surface Expression of GABAA Receptors 
Brain research  2010;1346:1-13.
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.
PMCID: PMC2941995  PMID: 20580636
GABAA receptor; γ-aminobutyric acid; ligand chaperone; endoplasmic reticulum; secretory pathway; GABA transporter; glutamic acid decarboxylase
21.  The benzodiazepine diazepam potentiates responses of α1β2γ2 γ-aminobutyric acid type A receptors activated by either γ-aminobutyric acid or allosteric agonists 
Anesthesiology  2013;118(6):10.1097/ALN.0b013e318289bcd3.
The γ-aminobutyric acid type A receptor is target for several anesthetics, anticonvulsants, anxiolytics and sedatives. Neurosteroids, barbiturates and etomidate both potentiate responses to γ-aminobutyric acid (GABA) and allosterically activate the receptor. We examined the ability of a benzodiazepine, diazepam, to potentiate responses to allosteric agonists.
The γ-aminobutyric acid type A receptors were expressed in human embryonic kidney 293 cells, and studied using whole-cell and single-channel patch clamp. The receptors were activated by the orthosteric agonist GABA, and allosteric agonists pentobarbital, etomidate and alfaxalone.
Diazepam is equally potent at enhancing responses to orthosteric and allosteric agonists. Diazepam EC50s were 25±4, 26±6, 33±6, and 26±3 nM for receptors activated by GABA, pentobarbital, etomidate, and alfaxalone, respectively (mean±S.D., 5–6 cells at each condition). Mutations to the benzodiazepine-binding site (α1(H101C), γ2(R144C), γ2(R197C)) reduced or removed potentiation for all agonists, and an inverse agonist at the benzodiazepine site reduced responses to all agonists. Single-channel data elicited by GABA demonstrate that in the presence of 1 μM diazepam the prevalence of the longest open-time component is increased from 13±7 (mean±S.D., n=5 patches) to 27±8 % (n=3 patches) and the rate of channel closing is decreased from 129±28 s−1 to 47±6 s−1 (mean±S.D.)
We conclude that benzodiazepines do not act by enhancing affinity of the orthosteric site for GABA but rather by increasing channel gating efficacy. The results also demonstrate the presence of significant interactions between allosteric activators and potentiators, raising a possibility of effects on dosage requirements or changes in side effects.
PMCID: PMC3852889  PMID: 23407108
22.  Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABAA Receptor Modulators? 
Despite decades of basic and clinical research, our understanding of how benzodiazepines tend to lose their efficacy over time (tolerance) is at least incomplete. In appears that tolerance develops relatively quickly for the sedative and anticonvulsant actions of benzodiazepines, whereas tolerance to anxiolytic and amnesic effects probably does not develop at all. In light of this evidence, we review the current evidence for the neuroadaptive mechanisms underlying benzodiazepine tolerance, including changes of (i) the GABAA receptor (subunit expression and receptor coupling), (ii) intracellular changes stemming from transcriptional and neurotrophic factors, (iii) ionotropic glutamate receptors, (iv) other neurotransmitters (serotonin, dopamine, and acetylcholine systems), and (v) the neurosteroid system. From the large variance in the studies, it appears that either different (simultaneous) tolerance mechanisms occur depending on the benzodiazepine effect, or that the tolerance-inducing mechanism depends on the activated GABAA receptor subtypes. Importantly, there is no convincing evidence that tolerance occurs with α subunit subtype-selective compounds acting at the benzodiazepine site.
PMCID: PMC3321276  PMID: 22536226
23.  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, GABAA receptors play a major role as targets for benzodiazepine drugs. The therapeutic relevance of the multitude of structurally diverse GABAA receptor subtypes has only recently been identified. α1-GABAA receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection of these drugs, whereas α2-GABAA receptors, but not α3-GABAA 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.
PMCID: PMC3181687  PMID: 22034214
GABA (γ-aminobutyric acid); GABAA receptor; neuronal inhibition; anxiety; epilepsy; schizophrenia; benzodiazepine
24.  Differential Roles of GABAA Receptor Subtypes in Benzodiazepine-Induced Enhancement of Brain-Stimulation Reward 
Neuropsychopharmacology  2012;37(11):2531-2540.
Benzodiazepines such as diazepam are widely prescribed as anxiolytics and sleep aids. Continued use of benzodiazepines, however, can lead to addiction in vulnerable individuals. Here, we investigate the neural mechanisms of the behavioral effects of benzodiazepines using the intracranial self-stimulation (ICSS) test, a procedure with which the reward-enhancing effects of these drugs can be measured. Benzodiazepines bind nonselectively to several different GABAA receptor subtypes. To elucidate the α subunit(s) responsible for the reward-enhancing effects of benzodiazepines, we examined mice carrying a histidine-to-arginine point mutation in the α1, α2, or α3 subunit, which renders the targeted subunit nonresponsive to diazepam, other benzodiazepines and zolpidem. In wild-type and α1-point-mutated mice, diazepam caused a dose-dependent reduction in ICSS thresholds (reflecting a reward-enhancing effect) that is comparable to the reduction observed following cocaine administration. This effect was abolished in α2- and α3-point-mutant mice, suggesting that these subunits are necessary for the reward-enhancing action of diazepam. α2 Subunits appear to be particularly important, since diazepam increased ICSS thresholds (reflecting an aversive-like effect) in α2-point-mutant animals. Zolpidem, an α1-preferring benzodiazepine-site agonist, had no reward-enhancing effects in any genotype. Our findings implicate α2 and α3 subunit containing GABAA receptors as key mediators of the reward-related effects of benzodiazepines. This finding has important implications for the development of new medications that retain the therapeutic effects of benzodiazepines but lack abuse liability.
PMCID: PMC3442348  PMID: 22763624
GABAA receptor; α subunit; benzodiazepine; intracranial self-stimulation; zolpidem; point mutation; a subunit; Addiction & Substance Abuse; benzodiazepine; GABA; GABAA receptor; intracranial self-stimulation; Neuropharmacology; Receptor Pharmacology; zolpidem
25.  Amygdala-specific reduction of α1-GABAA receptors disrupts the anticonvulsant, locomotor, and sedative, but not anxiolytic effects of benzodiazepines in mice 
The heterogeneity and distribution of GABAA receptor subunits mediates differential roles in behavior. It is thought that particular behavioral responses to benzodiazepine (BZ) ligands might be associated with an action at a regionally defined receptor subtype. However, the role of specific GABAA receptor subtypes in particular brain regions is less clear. Such detailed knowledge of regional α1-GABAA receptor function will advance our understanding of the neural circuitry underlying the role of GABAA receptors and the effects of GABAA-modulating drugs on behavior. By combining inducible, site-specific α1 subunit deletion, using a lentivirus expressing Cre-recombinase in mice with the α1 subunit gene flanked by loxP sites, we examine baseline and pharmacological effects of deletion of amygdala α1-GABAA receptors. We find that amygdala-specific reduction of α1 receptor subunits does not affect mRNA or protein levels of amygdala α2 or α3 subunit receptors. Nor does this inducible reduction affect baseline locomotion or measures of anxiety. However, we also find that this inducible, site-specific deletion does disrupt the normal sedative-locomotor inhibition as well as the anticonvulsive effects, of two distinct benzodiazepine-site ligands, diazepam and zolpidem, which is relatively α1-subunit selective. These data, utilizing inducible, region and subunit-specific deletion, combined with pharmacogenetic approaches, demonstrate that amygdala expression of the α1-GABAA receptor subunit is required for normal benzodiazepine effects on sedation, locomotion, and seizure inhibition, but not for anxiolysis.
PMCID: PMC2905594  PMID: 20505082

Results 1-25 (980006)