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Nature Jan 2019Type-A γ-aminobutyric (GABA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general...
Type-A γ-aminobutyric (GABA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA receptor modulators.
Topics: Allosteric Regulation; Alprazolam; Benzodiazepines; Bicuculline; Binding, Competitive; Cryoelectron Microscopy; Diazepam; GABA Modulators; Humans; Ligands; Models, Molecular; Nanostructures; Picrotoxin; Receptors, GABA-A; Signal Transduction
PubMed: 30602790
DOI: 10.1038/s41586-018-0832-5 -
The Journal of Experimental Medicine Dec 2022Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce...
Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce P53-dependent apoptosis in LGR5+ intestinal stem cells (ISCs). Gamma-aminobutyric acid (GABA) and its A receptors (GABAAR) are present in the gastrointestinal tract. However, the functioning of the GABAergic system in ISCs is poorly defined. We found that GABAAR α1 (GABRA1) levels increased in the murine intestine after chemoradiotherapy. GABRA1 depletion in LGR5+ ISCs protected the intestine from chemoradiotherapy-induced P53-dependent apoptosis and prolonged animal survival. The administration of bicuculline, a GABAAR antagonist, prevented chemoradiotherapy-induced ISC loss and intestinal damage without reducing the chemoradiosensitivity of tumors. Mechanistically, it was associated with the reduction of reactive oxygen species-induced DNA damage via the L-type voltage-dependent Ca2+ channels. Notably, flumazenil, a GABAAR antagonist approved by the U.S. Food and Drug Administration, rescued human colonic organoids from chemoradiotherapy-induced toxicity. Therefore, flumazenil may be a promising drug for reducing the gastrointestinal side effects of chemoradiotherapy.
Topics: Animals; Bicuculline; Calcium; Chemoradiotherapy; Flumazenil; Humans; Intestines; Mice; Reactive Oxygen Species; Receptors, GABA-A; Stem Cells; Tumor Suppressor Protein p53; United States; gamma-Aminobutyric Acid
PubMed: 36125780
DOI: 10.1084/jem.20220541 -
Neuron Nov 2023The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABARs)....
The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABARs). Pharmacological properties of ρ-type GABARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here, we present cryo-EM structures of a lipid-embedded human ρ1 GABAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of ρ1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to ρ-specific pharmaceutical design and to our biophysical understanding of ligand-gated ion channels.
Topics: Humans; Receptors, GABA-A; Picrotoxin; Ligands; gamma-Aminobutyric Acid; Bicuculline; Binding Sites
PubMed: 37659407
DOI: 10.1016/j.neuron.2023.08.006 -
Reviews in the Neurosciences Aug 2020In this review, a series of experiments is presented, in which γ-amino butyric acid (GABA)ergic and glutamatergic effects on dopamine function in the rat nigrostriatal... (Review)
Review
In this review, a series of experiments is presented, in which γ-amino butyric acid (GABA)ergic and glutamatergic effects on dopamine function in the rat nigrostriatal and mesolimbic system was systematically assessed after pharmacological challenge with GABAA receptor (R) and and N-methyl d-aspartate (NMDA)R agonists and antagonists. In these studies, [123I]iodobenzamide binding to the D2/3R was mesured in nucleus accumbens (NAC), caudateputamen (CP), substantia nigra/ventral tegmental area (SN/VTA), frontal (FC), motor (MC) and parietal cortex (PC) as well as anterior (aHIPP) and posterior hippocampus (pHIPP) with small animal SPECT in baseline and after injection of either the GABAAR agonist muscimol (1 mg/kg), the GABAAR antagonist bicuculline (1 mg/kg), the NMDAR agonist d-cycloserine (20 mg/kg) or the NMDAR antagonist amantadine (40 mg/kg). Muscimol reduced D2/3R binding in NAC, CP, SN/VTA, THAL and pHIPP, while, after amantadine, decreases were confined to NAC, CP and THAL. In contrast, d-cycloserine elevated D2/3R binding in NAC, SN/VTA, THAL, frontal cortex, motor cortex, PC, aHIPP and pHIPP, while, after bicuculline, increases were confined to CP and THAL. Taken together, similar actions on regional dopamine levels were exterted by the GABAAR agonist and the NMDAR antagonist on the one side and by the GABAAR antagonist and the NMDAR agonist on the other, with agonistic action, however, affecting more brain regions. Thereby, network analysis suggests different roles of GABAARs and NMDARs in the mediation of nigrostriatal, nigrothalamocortical and mesolimbocortical dopamine function.
Topics: Animals; Bicuculline; Dopamine; Humans; Muscimol; Nucleus Accumbens; Rats; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate
PubMed: 32619197
DOI: 10.1515/revneuro-2019-0112 -
Nature Communications Feb 2023Epilepsy is a disorder in which abnormal neuronal hyperexcitation causes several types of seizures. Because pharmacological and surgical treatments occasionally...
Epilepsy is a disorder in which abnormal neuronal hyperexcitation causes several types of seizures. Because pharmacological and surgical treatments occasionally interfere with normal brain function, a more focused and on-demand approach is desirable. Here we examined the efficacy of a chemogenetic tool-designer receptors exclusively activated by designer drugs (DREADDs)-for treating focal seizure in a nonhuman primate model. Acute infusion of the GABA receptor antagonist bicuculline into the forelimb region of unilateral primary motor cortex caused paroxysmal discharges with twitching and stiffening of the contralateral arm, followed by recurrent cortical discharges with hemi- and whole-body clonic seizures in two male macaque monkeys. Expression of an inhibitory DREADD (hM4Di) throughout the seizure focus, and subsequent on-demand administration of a DREADD-selective agonist, rapidly suppressed the wide-spread seizures. These results demonstrate the efficacy of DREADDs for attenuating cortical seizure in a nonhuman primate model.
Topics: Male; Animals; Seizures; Brain; Bicuculline; Body Fluids; GABA-A Receptor Antagonists; Macaca
PubMed: 36854724
DOI: 10.1038/s41467-023-36642-6 -
International Journal of Molecular... Nov 2022Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA...
Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl via the GABA receptor or efflux or K via the GABA receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABA and GABA receptors induces an increase in intracellular Ca and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABA receptor, as it was blunted by the GABA receptor antagonist bicuculline but unaffected by GABA receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.
Topics: Rats; Animals; Connexin 43; Astrocytes; Receptors, GABA-A; Bicuculline; Animals, Newborn; Cells, Cultured; Glutamic Acid; gamma-Aminobutyric Acid; Adenosine Triphosphate
PubMed: 36362410
DOI: 10.3390/ijms232113625 -
American Journal of Physiology.... Oct 2019Functional gastrointestinal disorders, including delayed gastric emptying and decreased gastric motility, are more prevalent in women, suggesting a potential role for...
Functional gastrointestinal disorders, including delayed gastric emptying and decreased gastric motility, are more prevalent in women, suggesting a potential role for circulating gonadal hormones, including estrogen. Gastric motility is tuned by the vagal inputs arising from the dorsal motor nucleus of the vagus (DMV), which is itself controlled by tonic GABAergic inputs. Estrogen increases GABA functions in various central nervous system areas; however, the effect of the estrus cycle in modulating GABAergic inputs onto DMV neurons, hence vagal control of gastric motility, has not been investigated. The aim of the present study was to test the hypothesis that GABAergic tone to DMV neurons, hence the vagal output to the stomach, varies according to sex and the estrus cycle. Experiments were performed on age-matched Sprague-Dawley male and virgin female rats; females were subdivided according to the high-estrogen (HE) or low-estrogen (LE) period of their cycle. Whole-cell patch-clamp recordings were made from gastric-projecting DMV neurons, and the response to perfusion with the GABA receptor antagonist bicuculline was examined. The response of corpus and antrum tone and motility to bicuculline microinjected in the dorsal vagal complex, recorded via strain gauges sewn to the anterior gastric surface, was also assessed. Bicuculline increased the firing rate of DMV neurons, as well as gastric tone and motility, to a larger extent in HE compared with LE or male rats, suggesting a higher GABAergic tone in HE female rats. Taken together, the data support the hypothesis that GABAergic tone to DMV neurons varies according to sex and estrus cycle. GABAergic neurotransmission to the dorsal motor nucleus of the vagus (DMV) plays a pivotal role in the modulation of gastric tone and motility. Gastric motility is reduced in women and may contribute to the higher incidence of functional gastrointestinal disorders. In the present study, we report that GABAergic tone to rat DMV neurons, hence vagal output to the stomach, varies according to sex and estrus cycle, and the GABAergic tone is increased during the high-estrogen period of the estrus cycle.
Topics: Animals; Bicuculline; Estrogens; Estrous Cycle; Female; GABA Antagonists; Gastrointestinal Motility; Male; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sex Characteristics; Stomach; Synaptic Transmission; Vagus Nerve; gamma-Aminobutyric Acid
PubMed: 31393788
DOI: 10.1152/ajpgi.00112.2019 -
The International Journal of... Feb 2022This study aimed to investigate the connection between the mutation of the transcription factor and impaired Ca-signaling, which reflects changes in neurotransmission...
OBJECTIVE
This study aimed to investigate the connection between the mutation of the transcription factor and impaired Ca-signaling, which reflects changes in neurotransmission in the cerebral cortex .
METHODS
We used mixed neuroglial cortical cell cultures derived from mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used.
RESULTS
It was found that cortical neurons isolated from homozygous () mice with the mutation demonstrate suppressed Ca signals in models of epileptiform activity . Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca oscillations occurring in all neurons of the network in response to Mg-free medium and bicuculline. But cortical neurons only single Ca pulses or attenuated Ca oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca oscillations to a Mg-free medium and bicuculline increases.
CONCLUSION
IL-10 restores neurotransmission in neuronal networks with the mutation by regulating the expression of genes encoding signaling proteins.
Topics: Animals; Bicuculline; Calcium; Cells, Cultured; Cerebral Cortex; Interleukin-10; Mice; N-Methylaspartate; Receptors, Glutamate; Transcription Factors; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
PubMed: 32727246
DOI: 10.1080/00207454.2020.1803305 -
The Journal of Physiology Dec 2019•Initiation of pathological synchronous events such as epileptic spikes and seizures is linked to the hyperexcitability of the neuronal network in both humans and...
KEY POINTS
•Initiation of pathological synchronous events such as epileptic spikes and seizures is linked to the hyperexcitability of the neuronal network in both humans and animals. •In the present study, we show that epileptiform interictal-like spikes and seizures emerged in human neocortical slices by blocking GABA receptors, following the disappearance of the spontaneously occurring synchronous population activity. •Large variability of temporally and spatially simple and complex spikes was generated by tissue from epileptic patients, whereas only simple events appeared in samples from non-epileptic patients. •Physiological population activity was associated with a moderate level of principal cell and interneuron firing, with a slight dominance of excitatory neuronal activity, whereas epileptiform events were mainly initiated by the synchronous and intense discharge of inhibitory cells. •These results help us to understand the role of excitatory and inhibitory neurons in synchrony-generating mechanisms, in both epileptic and non-epileptic conditions.
ABSTRACT
Understanding the role of different neuron types in synchrony generation is crucial for developing new therapies aiming to prevent hypersynchronous events such as epileptic seizures. Paroxysmal activity was linked to hyperexcitability and to bursting behaviour of pyramidal cells in animals. Human data suggested a leading role of either principal cells or interneurons, depending on the seizure morphology. In the present study, we aimed to uncover the role of excitatory and inhibitory processes in synchrony generation by analysing the activity of clustered single neurons during physiological and epileptiform synchronies in human neocortical slices. Spontaneous population activity was detected with a 24-channel laminar microelectrode in tissue derived from patients with or without preoperative clinical manifestations of epilepsy. This population activity disappeared by blocking GABA receptors, and several variations of spatially and temporally simple or complex interictal-like spikes emerged in epileptic tissue, whereas peritumoural slices generated only simple spikes. Around one-half of the clustered neurons participated with an elevated firing rate in physiological synchronies with a slight dominance of excitatory cells. By contrast, more than 90% of the neurons contributed to interictal-like spikes and seizures, and an intense and synchronous discharge of inhibitory neurons was associated with the start of these events. Intrinsically bursting principal cells fired later than other neurons. Our data suggest that a balanced excitation and inhibition characterized physiological synchronies, whereas disinhibition-induced epileptiform events were initiated mainly by non-synaptically synchronized inhibitory neurons. Our results further highlight the differences between humans and animal models, and between in vivo and (pharmacologically manipulated) in vitro conditions.
Topics: Adult; Aged; Bicuculline; Epilepsy; Female; GABA-A Receptor Antagonists; Humans; Male; Middle Aged; Neocortex; Neurons; Receptors, GABA-A; Young Adult
PubMed: 31523807
DOI: 10.1113/JP278499 -
Neuroscience Research Nov 2022Altered inhibition/excitation (I/E) balance contributes to various brain disorders. Dysfunctional GABAergic interneurons enhance or reduce inhibition, resulting in I/E...
Altered inhibition/excitation (I/E) balance contributes to various brain disorders. Dysfunctional GABAergic interneurons enhance or reduce inhibition, resulting in I/E imbalances. Differences in short-term plasticity between excitation and inhibition cause frequency-dependence of the I/E ratio, which can be altered by GABAergic dysfunction. However, it is unknown whether I/E imbalances can be rescued pharmacologically using a single dose when the imbalance magnitude is frequency-dependent. Loss of PGC-1α (peroxisome proliferator activated receptor γ coactivator 1α) causes transcriptional dysregulation in hippocampal GABAergic interneurons. PGC-1α slices have enhanced baseline inhibition onto CA1 pyramidal cells, causing increased I/E ratio and impaired circuit function. High frequency stimulation reduces the I/E ratio and recovers circuit function in PGC-1α slices. Here we tested if using a low dose of bicuculline that can restore baseline I/E ratio can also rescue the frequency-dependent I/E imbalances in these mice. Remarkably, bicuculline did not reduce the I/E ratio below that of wild type during high frequency stimulation. Interestingly, bicuculline enhanced the paired-pulse ratio (PPR) of disynaptic inhibition without changing the monosynaptic inhibition PPR, suggesting that bicuculline modifies interneuron recruitment and not GABA release. Bicuculline improved CA1 output in PGC-1α slices, enhancing EPSP-spike coupling to wild type levels at high and low frequencies. Our results show that it is possible to rescue frequency-dependent I/E imbalances in an animal model of transcriptional dysregulation with a single treatment.
Topics: Animals; Bicuculline; Hippocampus; Interneurons; Mice; Mice, Knockout; PPAR gamma; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha
PubMed: 35842011
DOI: 10.1016/j.neures.2022.07.003