Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

GABAA receptor signalling mechanisms revealed by structural pharmacology.

Type-A γ-aminobutyric (GABAA) 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 GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1ß3γ2L GABAA 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 GABAA 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 GABAA receptor modulators.

The role of HCN channels on the effects of T-type calcium channels and GABAA receptors in the absence epilepsy model of WAG/Rij rats.

In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.

Optimizing GABAA receptor antagonism for the induction of long-term potentiation in the mouse and rat dentate gyrus in vitro.

The reliable induction of long-term potentiation (LTP) in the dentate gyrus (DG) in vitro requires the blockade of the γ-aminobutyric acid A (GABAA) receptor. In these studies we examined the effectiveness of the specific GABAA receptor antagonist bicuculline methiodide (BMI) in facilitating LTP in the DG from hippocampal slices obtained from either C57Bl/6 mice or Sprague-Dawley rats, two species commonly used for electrophysiology. In the C57Bl/6 mice, maximal short-term potentiation and LTP in the DG were produced with a concentration of 5 µM BMI. In contrast, a concentration of 10 µM BMI was required to produce maximal short-term potentiation and LTP in the DG of Sprague-Dawley rats. These results reveal that there are species differences in the optimal amount of BMI required to produce robust and reliable LTP in the rodent DG in vitro and highlight the need to take consideration of the species being used when choosing concentrations of pharmacological agents to employ for electrophysiological use.NEW & NOTEWORTHY In this report we provide specific neurophysiological evidence for concentrations of GABAA antagonist required to study long-term potentiation in the medial perforant pathway of the dentate gyrus. Two commonly used species, Sprague-Dawley rats and C57Bl/6 mice, require different concentrations of bicuculline methiodide to induce optimal short-term and long-term potentiation.

Additive effect of histamine and muscimol upon induction of antinociceptive and antidepressant effects in mice.

We investigated the effects of histamine and GABA A receptor agents on pain and depression-like behaviors and their interaction using a tail-flick test and the forced swimming test (FST) in male mice. Our data revealed that intraperitoneal administration of muscimol (0.12 and 0.25 mg/kg) increased the percentage of maximum possible effect (%MPE) and area under the curve (AUC) of %MPE, indicating an antinociceptive response. Intraperitoneal injection of bicuculline (0.5 and 1 mg/kg) decreased %MPE and AUC of %MPE, suggesting hyperalgesia. Moreover, muscimol by reducing the immobility time of the FST elicited an antidepressant-like response but bicuculline by enhancing the immobility time of the FST caused a depressant-like response. Intracerebroventricular (i.c.v.) microinjection of histamine (5 µg/mouse) enhanced %MPE and AUC of %MPE. i.c.v. infusion of histamine (2.5 and 5 µg/mouse) decreased immobility time in the FST. Co-administration of different doses of histamine along with a sub-threshold dose of muscimol potentiated antinociceptive and antidepressant-like responses produced by histamine. Cotreatment of different doses of histamine plus a noneffective dose of bicuculline reversed antinociception and antidepressant-like effects elicited by histamine. Cotreatment of histamine, muscimol, and bicuculline reversed antinociceptive and antidepressant-like behaviors induced by the drugs. The results demonstrated additive antinociceptive and antidepressant-like effects between histamine and muscimol in mice. In conclusion, our results indicated an interaction between the histaminergic and GABAergic systems in the modulation of pain and depression-like behaviors.

Structure of a human synaptic GABAA receptor.

Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABAA receptor). Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABAA receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1ß2γ2 GABAA receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.

Pharmacokinetics, tissue distribution, and plasma protein binding ratio of bicuculline following intragastric and intravenous administration in rats using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Bicuculline is a natural isoquinoline alkaloid that works as a gamma-aminobutyric acid receptor antagonist. It is widely found in Papaveraceae plants used in traditional Chinese medicines. Bicuculline not only has been shown to have favorable analgesic, memory-improving, and anxiolytic effects but may also cause adverse effects such as convulsions and epilepsy. A simple, rapid, and sensitive method was developed and validated for the determination of bicuculline in the plasma and tissue samples in rats by ultra-high-performance liquid chromatography-tandem mass spectrometry (MS/MS). The chromatographic separation was performed on a Thermo Scientific C18 column. The MS/MS system was operated in the positive multiple reaction monitoring mode, and the precursor-product ion transitions were optimized as m/z 368.0 → 307.1 for bicuculline and as 354.1 → 188.1 for protopine (internal standard). The linearity, accuracy, precision, recovery, and matrix effect were within acceptable limits. The experimental data showed that bicuculline was rapidly absorbed and eliminated in rats, with a moderate plasma protein binding ratio and low bioavailability. The main tissues of distribution were the kidney, liver, and brain; bicuculline could exert its pharmacological effects across the blood-brain barrier. This study has positive implications for the clinical use of herbal medicines containing bicuculline and for further development.

Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus.

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

Excitatory-inhibitory mismatch shapes node recruitment in an epileptic network.

OBJECTIVE: Focal epilepsy is thought to be a network disease, in which epileptiform activity can spread noncontiguously through the brain via highly interconnected nodes, or hubs, within existing networks. Animal models confirming this hypothesis are scarce, and our understanding of how distant nodes are recruited is also lacking. Whether interictal spikes (IISs) also create and reverberate through a network is not well understood. METHODS: We injected bicuculline into the S1 barrel cortex and employed multisite local field potential and Thy-1 and parvalbumin (PV) cell mesoscopic calcium imaging during IISs to monitor excitatory and inhibitory cells in two monosynaptically connected nodes and one disynaptically connected node: ipsilateral secondary motor area (iM2), contralateral S1 (cS1), and contralateral secondary motor area (cM2). Node participation was analyzed with spike-triggered coactivity maps. Experiments were repeated with 4-aminopyridine as an epileptic agent. RESULTS: We found that each IIS reverberated throughout the network, differentially recruiting both excitatory and inhibitory cells in all connected nodes. The strongest response was found in iM2. Paradoxically, node cM2, which was connected disynaptically to the focus, was recruited more intensely than node cS1, which was connected monosynaptically. The explanation for this effect could be found in node-specific excitatory/inhibitory (E/I) balance, as cS1 demonstrated greater PV inhibitory cell activation compared with cM2, where Thy-1 excitatory cells were more heavily recruited. SIGNIFICANCE: Our data show that IISs spread noncontiguously by exploiting fiber pathways that connect nodes in a distributed network and that E/I balance plays a critical role in node recruitment. This multinodal IIS network model can be used to investigate cell-specific dynamics in the spatial propagation of epileptiform activity.

Adenosine and γ-aminobutyric acid partially regulate metabolic and ventilatory responses of Damaraland mole-rats to acute hypoxia.

Fossorial Damaraland mole-rats (Fukomys damarensis) mount a robust hypoxic metabolic response (HMR) but a blunted hypoxic ventilatory response (HVR) to acute hypoxia. Although these reflex physiological responses have been described previously, the underlying signalling pathways are entirely unknown. Of particular interest are contributions from γ-aminobutyric acid (GABA), which is the primary inhibitory neurotransmitter in the nervous system of most adult mammals, and adenosine, the accumulation of which increases during hypoxia as a breakdown product of ATP. Therefore, we hypothesized that GABAergic and/or adenosinergic signalling contributes to the blunted HVR and robust HMR in Damaraland mole-rats. To test this hypothesis, we injected adult animals with saline alone (controls), or 100 mg kg-1 aminophylline or 1 mg kg-1 bicuculline, to block adenosine or GABAA receptors, respectively. We then used respirometry, plethysmography and thermal RFID probes to non-invasively measure metabolic, ventilator and thermoregulatory responses, respectively, to acute hypoxia (1 h in 5 or 7% O2) in awake and freely behaving animals. We found that bicuculline had relatively minor effects on metabolism and thermoregulation but sensitized ventilation such that the HVR became manifest at 7% instead of 5% O2 and was greater in magnitude. Aminophylline increased metabolic rate, ventilation and body temperature in normoxia, and augmented the HMR and HVR. Taken together, these findings indicate that adenosinergic and GABAergic signalling play important roles in mediating the robust HMR and blunted HVR in Damaraland mole-rats.

Pharmacological evaluation of lateral habenula and rostromedial tegmental nucleus in the expression of ethanol-induced place preference.

Although ethanol administration produces a range of physiological effects, the rewarding aspect associated with its consumption is a major contributory factor to its abuse liability. Recently, lateral habenula (LHb) has been shown to be engaged by both rewarding and aversive stimuli. Its major glutamatergic output, the fasciculus retroflexus, projects to the rostromedial tegmental nucleus (RMTg) and controls the activity of the ventral tegmental area (VTA) dopaminergic system to promote reward circuitry. While several attempts have been made to understand the relationship between LHb and addiction, there is still a lack of knowledge in relation to ethanol addiction. In the present study, by pharmacologically exacerbating or inhibiting the LHb or RMTg neuronal activity during a post-conditioning test, we investigated the role of LHb-RMTg fasciculus retroflexus in ethanol-induced reward behavior using the conditioned place preference (CPP) test. We found that activation of LHb glutamatergic system by intra-LHb administration of l-trans-2,4-pyrrolidine dicarboxylate (PDC) (glutamate transporter inhibitor) significantly decreased CPP score; on the contrary, lamotrigine (inhibits glutamate release) significantly increased CPP score and showed a rewarding effect in CPP. Instead, intra-RMTg administration of muscimol (GABAA receptor agonist) significantly increased CPP score, whereas bicuculline (GABAA antagonist) treatment decreased CPP score. In immunohistochemistry, we found that PDC administration significantly decreased, whereas lamotrigine treatment significantly increased tyrosine hydroxylase immunoreactivity (TH-ir) in VTA and nucleus accumbens (NAc). Furthermore, while intra-RMTg administration of muscimol increased, the bicuculline treatment significantly decreased the TH-ir in VTA and NAc. Together, our behavioral and immunohistochemical results signify the role of LHb and RMTg in the expression of ethanol-conditioned reward behavior.

Origin of Retinal Oscillatory Potentials in the Mouse, a Tool to Specifically Locate Retinal Damage.

To determine the origin of oscillatory potentials (OPs), binocular electroretinogram (ERG) recordings were performed under light and dark adaptation on adult healthy C57BL/6J mice. In the experimental group, 1 µL of PBS was injected into the left eye, while the right eye was injected with 1 µL of PBS containing different agents: APB, GABA, Bicuculline, TPMPA, Glutamate, DNQX, Glycine, Strychnine, or HEPES. The OP response depends on the type of photoreceptors involved, showing their maximum response amplitude in the ERG induced by mixed rod/cone stimulation. The oscillatory components of the OPs were affected by the injected agents, with some drugs inducing the complete abolition of oscillations (APB, GABA, Glutamate, or DNQX), whereas other drugs merely reduced the oscillatory amplitudes (Bicuculline, Glycine, Strychnine, or HEPES) or did not even affect the oscillations (TPMPA). Assuming that rod bipolar cells (RBC) express metabotropic Glutamate receptors, GABAA, GABAC, and Glycine receptors and that they release glutamate mainly on Glycinergic AII amacrine cells and GABAergic A17 amacrine cells, which are differently affected by the mentioned drugs, we propose that RBC-AII/A17 reciprocal synapses are responsible for the OP generation in the ERG recordings in the mice. We conclude that the reciprocal synapses between RBC and AII/A17 are the basis of the ERG OP oscillations of the light response, and this fact must be taken into consideration in any ERG test that shows a decrease in the OPs' amplitude.

Of the Mechanisms of Paroxysmal Depolarization Shifts: Generation and Maintenance of Bicuculline-Induced Paroxysmal Activity in Rat Hippocampal Cell Cultures.

Abnormal depolarization of neuronal membranes called paroxysmal depolarization shift (PDS) represents a cellular correlate of interictal spikes. The mechanisms underlying the generation of PDSs or PDS clusters remain obscure. This study aimed to investigate the role of ionotropic glutamate receptors (iGluRs) in the generation of PDS and dependence of the PDS pattern on neuronal membrane potential. We have shown that significant depolarization or hyperpolarization (by more than ±50 mV) of a single neuron does not change the number of individual PDSs in the cluster, indicating the involvement of an external stimulus in PDS induction. Based on this data, we have suggested reliable protocols for stimulating single PDS or PDS clusters. Furthermore, we have found that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are necessary for PDS generation since AMPAR antagonist NBQX completely suppresses bicuculline-induced paroxysmal activity. In turn, antagonists of NMDA (N-methyl-D-aspartate) and kainate receptors (D-AP5 and UBP310, respectively) caused a decrease in the amplitude of the first action potential in PDSs and in the amplitude of the oscillations of intracellular Ca2+ concentration occurring alongside the PDS cluster generation. The effects of the NMDAR (NMDA receptor) and KAR (kainate receptor) antagonists indicate that these receptors are involved only in the modulation of paroxysmal activity. We have also shown that agonists of some Gi-coupled receptors, such as A1 adenosine (A1Rs) or cannabinoid receptors (CBRs) (N6-cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A1R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on Gi-coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy.

Presynaptic nigral GPR55 receptors stimulate [3 H]-GABA release through [3 H]-cAMP production and PKA activation and promote motor behavior.

Striatal medium-sized spiny neurons express mRNA and protein of GPR55 receptors that stimulate neurotransmitter release; thus, GPR55 could be sent to nigral striatal projections, where it might modulate GABA release and motor behavior. Here, we study the presence of GPR55 receptors at striato-nigral terminals, their modulation of GABA release, their signaling pathway, and their effect on motor activity. By double immunohistochemistry, we found the colocation of GPR55 protein and substance P in the dorsal striatum. In slices of the rat substantia nigra, the GPR55 agonists LPI and O-1602 stimulated [3 H]-GABA release induced by high K+ depolarization in a dose-dependent manner. The antagonists CID16020046 and cannabidiol prevented agonist stimulation in a dose-dependent way. The effect of GPR55 on nigral [3 H]-GABA release was prevented by lesion of the striatum with kainic acid, which was accompanied by a decrement of GPR55 protein in nigral synaptosomes, indicating the presynaptic location of receptors. The depletion of internal Ca2+ stores with thapsigargin did not prevent the effect of LPI on [3 H]-GABA release, but the remotion or chelation of external calcium did. Blockade of Gi, Gs, PLC, PKC, or dopamine D1 receptor signaling proteins did not prevent the effect of GPR55 on release. However, the activation of GPR55 stimulated [3 H]-cAMP accumulation and PKA activity. Intranigral unilateral injection of LPI induces contralateral turning. This turning was prevented by CID16020046, cannabidiol, and bicuculline but not by SCH 23390. Our data indicate that presynaptic GPR55 receptors stimulate [3 H]-GABA release at striato-nigral terminals through [3 H]-cAMP production and stimulate motor behavior.

Involvement of GABAA receptors of lateral habenula in the acquisition and expression phases of morphine-induced place preference in male rats.

The lateral habenula (LHb) is a critical brain structure involved in the aversive response to drug abuse. It has been determined that the gamma-aminobutyric acid (GABA)-ergic system plays the main role in morphine dependency. The role of GABA type A receptors (GABAARs) in LHb on morphine-induced conditioned place preference (CPP) remains unknown. In this study, the effect of bilateral intra-LHb microinjection of GABAAR agonist and antagonist on the acquisition and expression phases of CPP, utilizing a 5-day CPP paradigm in male rats, was evaluated. Subcutaneous administration of different doses of morphine caused a dose-dependent CPP. Intra-LHb microinjection of the GABAAR agonist, muscimol, in combination with morphine (5 mg/kg; subcutaneously) enhanced CPP scores in the acquisition phase of morphine CPP, whereas the GABAAR antagonist, bicuculline, significantly reduced the conditioning scores in the acquisition phase. Furthermore, pretreatment with a high dose of bicuculline reversed the additive effect of muscimol during the acquisition phase, yet the low dose of antagonist had no significant effect on agonist-induced CPP scores. On the other hand, muscimol (3 µg/rat) significantly increased CPP scores in the expression phase but bicuculline did not induce a significant effect on CPP scores. Bicuculline and muscimol microinjections did not affect locomotor activity in the testing sessions. Our results confirm that GABAARs in LHb play an active role in morphine reward. In addition, microinjections of bicuculline/muscimol may alter the morphine response through the GABAergic system.

Punicalagin and ellagic acid containing Punica granatum L. fruit rind extract prevents vincristine-induced neuropathic pain in rats: an in silico and in vivo evidence of GABAergic action and cytokine inhibition.

Objectives: We aimed to investigate the protective potential of Punica granatum L. fruit rind extract (PFE) containing punicalagin (10.3% W/W), ellagic acid (EA) (2.7%W/W) in vincristine (75 µg/kg i.p.)- induced neuropathic pain in Wistar rats.Methods: Docking simulation studies were done on the three-dimensional (3D) structure of the GABAA and PPAR γ receptor for the binding of EA as well as punicalagin docking studies on TNF-α, and IL-6. The Present Study conceptualized a test battery to evaluate the behavioral, biochemical and histological changes.Results: Vincristine -induced significant cold allodynia, mechanical hyperalgesia, and functional deficit on 12th and 21st days. It also increased in the levels of TNF-α (Tumor necrosis factor-α), IL-6 (Interleukin-6), and MPO (Myeloperoxidase). Administration of PFE (100 and 300 mg/kg, p.o.), EA (50 mg/kg), and gabapentin (100 mg/kg) attenuated Vincristine-induced behavioral and biochemical changes significantly (P < .05). PFE showed better antinociceptive activity to EA. The histopathological evaluation also revealed the protective effects of PFE. Pretreatment of bicuculline (selective antagonist of GABAA receptors) reversed antinociceptive action of PFE, but administration of γ aminobutyric acid potentiated the action of PFE. PPAR-γ antagonist BADGE did not modify the effect of PFE. Docking results revealed that EA properly positioned into GABA and PPARγ binding site and acts as a partial agonist. Docking score of Punicalagin found to be - 9.02 kcal/mol and - 8.32 kcal/mol on IL-6 and TNFα respectively.Discussion: Conclusively, the attenuating effect of PFE may be attributed to the GABAergic system, cytokine inhibition, and anti-inflammatory activities.

Interleukin-10 restores glutamate receptor-mediated Ca2+-signaling in brain circuits under loss of Sip1 transcription factor.

OBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 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 (Sip1fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1fl/fl 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 Sip1fl/fl 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 Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

Relative Contribution of Blood Pressure and Renal Sympathetic Nerve Activity to Proximal Tubular Sodium Reabsorption via NHE3 Activity.

We examined the effects of an acute increase in blood pressure (BP) and renal sympathetic nerve activity (rSNA) induced by bicuculline (Bic) injection in the paraventricular nucleus of hypothalamus (PVN) or the effects of a selective increase in rSNA induced by renal nerve stimulation (RNS) on the renal excretion of sodium and water and its effect on sodium-hydrogen exchanger 3 (NHE3) activity. Uninephrectomized anesthetized male Wistar rats were divided into three groups: (1) Sham; (2) Bic PVN: (3) RNS + Bic injection into the PVN. BP and rSNA were recorded, and urine was collected prior and after the interventions in all groups. RNS decreased sodium (58%) and water excretion (53%) independently of BP changes (p < 0.05). However, after Bic injection in the PVN during RNS stimulation, the BP and rSNA increased by 30% and 60% (p < 0.05), respectively, diuresis (5-fold) and natriuresis (2.3-fold) were increased (p < 0.05), and NHE3 activity was significantly reduced, independently of glomerular filtration rate changes. Thus, an acute increase in the BP overcomes RNS, leading to diuresis, natriuresis, and NHE3 activity inhibition.

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels.

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 GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-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 Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB 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.

Inhibition of GABAAR or Application of Lactobacillus casei Zhang Alleviates Ulcerative Colitis in Mice: GABAAR as a Potential Target for Intestinal Epithelial Renewal and Repair.

Emerging evidence indicates that the gamma-aminobutyric acid type A receptor (GABAAR) and Lactobacillus casei Zhang regulate colitis in a variety of ways, such as by participating in host immune and inflammatory responses, altering the gut microbiota, and influencing intestinal barrier function. However, not much is known about the mechanisms by which GABAAR and L. casei affect colon epithelial cell renewal and the interaction between GABAAR and L. casei during this process. To elucidate this, we established a dextran sulfate sodium (DSS)-induced model and measured the mouse body weights, colon length, the disease activity index (DAI), and histological scores. Our results indicated that inhibition of GABAAR alleviated the DSS-induced colitis symptoms, resulting in less weight loss and more intact colon tissue. Moreover, treatment with bicuculline (Bic, a GABAAR inhibitor) increased the levels of PCNA, ß-catenin, and TCF4 in mice with colitis. Interestingly, open field test performances showed that inhibition of GABAAR also attenuated colitis-related anxiety-like behavior. By 16S RNA gene sequencing analysis, we showed that inhibition of GABAAR partially reversed the gut dysbacteriosis of DSS-induced mice and increased the abundance of beneficial bacteria. Additionally, L. casei Zhang supplementation inhibited the expression of GABAAR in mice with colitis, promoted the proliferation and renewal of colon epithelial cells, and alleviated anxiety-like behavior and intestinal microflora disorder in mice. Thus, GABAAR plays a key role in the beneficial effects of L. casei on DSS-induced colitis in mice.