Presence Of GABA Research Articles

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

Identifying GABA-A Receptor Modulators that Bind to Intersubunit Sites in the GABA-A Receptor Transmembrane Domain

GABAARs, the major inhibitory neurotransmitter receptors in the brain, belong to the superfamily of pentameric ligand-gated ion channels, each containing five identical/homologous subunits arranged pseudosymmetrically around a central ion channel. For α1β3γ2 GABAARs, the most common subtype, subunits are ordered βαβαγ, with transmitter binding sites at the β+-α- interfaces in the extracellular domain. Two homologous but pharmacologically distinct classes of general anesthetic binding sites have been identified in the GABAAR transmembrane domain by use of photoreactive general anesthetics. In a α1β3γ2 GABAAR, azietomidate binds with high affinity (IC50 = 3 μM) and 100-fold selectivity to β+-α- sites, while a mephobarbital analog, R-mTFD-MPAB, binds to α+-β-/γ+-β- sites (IC50 = 1.3 μM) with 60-fold selectivity. Competition photolabeling assays using these photoprobes have identified the selectivity and affinities of other general anesthetics for these sites. Now, extending this approach, we characterized competitive and allosteric interactions of non-anesthetic GABAAR modulators with these sites. Ivermectin, a GABAAR potentiator known to bind subunit interface sites in homologous receptors, is ∼100-fold selective for R-mTFD-MPAB sites (IC50 = 20 nM) compared to azietomidate sites (IC50 = 2 μM). In the presence of GABA, loreclezole, an anti-epileptic agent and GABAAR potentiator, binds with highest affinity to the α+-β- site (IC50 = 1 µM), intermediate affinity to the β+-α- sites (IC50 = 10 µM), and low affinity to the γ+-β- site (IC50 = 230 µM). This photolabeling assay was also used to identify allosteric interactions between GABAAR binding sites. Picrotoxinin, a GABAAR channel blocker, is a potent allosteric modulator of R-mTFD-MPAB binding in the presence of bicuculline (IC50 = 0.5 μM), but it is >200-fold weaker (IC50 = 120 μM) in the presence of GABA and has no effect on azietomidate binding. Supported by GM58448.

Analysis of γ-Aminobutyric Acid Content in Fermented Plant Products by HPLC/UV

-Aminobutyric acid (GABA) content in fermented plant products and their main plant materials (aerial part of Acanthopanax sessiliflorus, fruit of Crataegus pinnatifida, and whole plant of Morus alba) was determined by high-performance liquid chromatography. GABA was quantified using a reverse-phase column with a gradient elution program (water:acetonitrile =90:10 to 0:100 for 40 min). UV detection was conducted at 280 nm. GABA content was measured in fermented plant products (15.07 mg/g), aerial part of A. sessiliflorus (4.49 mg/g), fruit of C. pinnatifida (10.59 mg/g), and whole plant of M. alba (2.31 mg/g). The presence of GABA in fermented plant products, including A. sessiliflorus, C. pinnatifida, and M. alba is important in industrial application for health supplements.

Insect Herbivory-Elicited GABA Accumulation in Plants is a Wound-Induced, Direct, Systemic, and Jasmonate-Independent Defense Response.

The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all organisms analyzed so far. In invertebrates GABA acts as a neurotransmitter; in plants different functions are under discussion. Among others, its involvement in abiotic stress reactions and as a defensive compound against feeding insects is suggested. GABA is synthesized from glutamate by glutamate decarboxylases and degraded by GABA-transaminases. Here, in Arabidopsis thaliana, gad1/2 double mutants showing reduced GABA concentrations as well as GABA-enriched triple mutants (gad1/2 x pop2-5) were generated and employed for a systematic study of GABA induction, accumulation and related effects in Arabidopsis leaves upon herbivory. The results demonstrate that GABA accumulation is stimulated by insect feeding-like wounding by a robotic caterpillar, MecWorm, as well as by real insect (Spodoptera littoralis) herbivory. Higher GABA levels in both plant tissue and artificial dietary supplements in turn affect the performance of feeding larvae. GABA enrichment occurs not only in the challenged but also in adjacent leaf. This induced response is neither dependent on herbivore defense-related phytohormones, jasmonates, nor is jasmonate induction dependent on the presence of GABA. Thus, in Arabidopsis the rapid accumulation of GABA very likely represents a general, direct and systemic defense reaction against insect herbivores.

To Break or to Brake Neuronal Network Accelerated by Ammonium Ions?

PurposeThe aim of present study was to investigate the effects of ammonium ions on in vitro neuronal network activity and to search alternative methods of acute ammonia neurotoxicity prevention.MethodsRat hippocampal neuronal and astrocytes co-cultures in vitro, fluorescent microscopy and perforated patch clamp were used to monitor the changes in intracellular Ca2+- and membrane potential produced by ammonium ions and various modulators in the cells implicated in neural networks.ResultsLow concentrations of NH4Cl (0.1–4 mM) produce short temporal effects on network activity. Application of 5–8 mM NH4Cl: invariably transforms diverse network firing regimen to identical burst patterns, characterized by substantial neuronal membrane depolarization at plateau phase of potential and high-amplitude Ca2+-oscillations; raises frequency and average for period of oscillations Ca2+-level in all cells implicated in network; results in the appearance of group of «run out» cells with high intracellular Ca2+ and steadily diminished amplitudes of oscillations; increases astrocyte Ca2+-signalling, characterized by the appearance of groups of cells with increased intracellular Ca2+-level and/or chaotic Ca2+-oscillations. Accelerated network activity may be suppressed by the blockade of NMDA or AMPA/kainate-receptors or by overactivation of AMPA/kainite-receptors. Ammonia still activate neuronal firing in the presence of GABA(A) receptors antagonist bicuculline, indicating that «disinhibition phenomenon» is not implicated in the mechanisms of networks acceleration. Network activity may also be slowed down by glycine, agonists of metabotropic inhibitory receptors, betaine, L-carnitine, L-arginine, etc.ConclusionsObtained results demonstrate that ammonium ions accelerate neuronal networks firing, implicating ionotropic glutamate receptors, having preserved the activities of group of inhibitory ionotropic and metabotropic receptors. This may mean, that ammonia neurotoxicity might be prevented by the activation of various inhibitory receptors (i.e. by the reinforcement of negative feedback control), instead of application of various enzyme inhibitors and receptor antagonists (breaking of neural, metabolic and signaling systems).

Bud extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing through GABAA and benzodiazepine receptors activation

Ethnopharmacological relevanceTilia tomentosa Moench bud extracts (TTBEs) is used in traditional medicine for centuries as sedative compound. Different plants belonging to the Tilia genus have shown their efficacy in the treatment of anxiety but still little is known about the mechanism of action of their bud extracts. Aim of the studyTo evaluate the action of TTBEs as anxiolytic and sedative compound on in vitro hippocampal neurons. Material and methodsThe anxiolytic effect of TTBEs was assayed by testing the effects of these compounds on GABAA receptor-activated chloride current of hippocampal neurons by means of the patch-clamp technique and microelectrode-arrays (MEAs). ResultsTTBEs acutely administered on mouse hippocampal neurons, activated a chloride current comparable to that measured in the presence of GABA (100µM). Bicuculline (100µM) and picrotoxin (100µM) blocked about 90% of this current, while the remaining 10% was blocked by adding the benzodiazepine (BDZ) antagonist flumazenil (30µM). Flumazenil alone blocked nearly 60% of the TTBEs activated current, suggesting that TTBEs binds to both GABAA and BDZ receptor sites. Application of high-doses of TTBEs on spontaneous active hippocampal neurons grown for 3 weeks on MEAs blocked the synchronous activity of these neurons. The effects were mimicked by GABA and prevented by picrotoxin (100µM) and flumazenil (30µM). At minimal doses, TTBEs reduced the frequency of synchronized bursts and increased the cross-correlation index of synchronized neuronal firing. ConclusionsOur data suggest that TTBEs mimics GABA and BDZ agonists by targeting hippocampal GABAergic synapses and inhibiting network excitability by increasing the strength of inhibitory synaptic outputs. Our results contribute toward the validation of TTBEs as effective sedative and anxiolytic compound.

Regional modulation of the response to glutathione in Hydra vulgaris.

In the presence of prey, or upon exposure to reduced glutathione (GSH), Hydra polyps open a mouth to ingest the captured prey and close it after feeding; at rest the mouth is not evident. In previous papers we have shown that GABA, glycine and NMDA modulate the mechanisms of mouth closure through ligand-gated-ion-channel receptors that are similar to their mammalian analogues in terms of biochemical and pharmacological properties. In order to study the regional distribution of these receptors, we have applied the GSH assay to polyps amputated at different levels of the body column. The response to 1-10 µmoll(-1) GSH of polyps lacking either peduncle and foot or the entire body columns (heads) was not different from control, whole animals. In the presence of GABA or muscimol, duration of the response was significantly decreased in heads; the decrease was suppressed by the GABA antagonists gabazine and bicuculline. By contrast, in animals lacking peduncle and foot, duration of the response did not vary upon GABA administration. Conversely, in the presence of glycine, duration of the response in heads preparations was similar to control, whereas in footless polyps, it was significantly reduced. The decrease was mimicked by the glycine agonists taurine and β-alanine, and counteracted by strychnine. These results suggest a regional distribution of receptors to GABA and glycine in the neuromuscular circuitry modulating the feeding behaviour.

Evaluation of peripheral versus central effects of GABA(B) receptor activation using a novel, positive allosteric modulator of the GABA(B) receptor ADX71943, a pharmacological tool compound with a fully peripheral activity profile.

The GABA(B) receptor agonist, baclofen, has shown promising effects in patients suffering from pain, post-traumatic stress disorder, alcoholism, overactive bladder and gastroesophageal reflux disease. However, baclofen's short duration of action and side effects limit its wider use. Here we characterized a novel, GABA(B) receptor positive allosteric modulator (PAM) ADX71943. In vitro, ADX71943 was assessed for pharmacological activity and selectivity using recombinant and native GABA(B) receptors. In vivo ADX71943 was assessed in the acetic acid-induced writhing (AAW) test in mice and formalin tests (FTs) in mice and rats. Marble burying (MB) and elevated plus maze (EPM) tests, rotarod, spontaneous locomotor activity (sLMA) and body temperature (BT) tests in mice and rats were used to investigate centrally-mediated effects. In vitro, in the presence of GABA, ADX71943 increased the potency and efficacy of agonists and showed selectivity at the GABA(B) receptor. ADX71943 reduced pain-associated behaviours in AAW; an effect blocked by GABA(B) receptor antagonist CGP63360. ADX71943 reduced pain in the FT in mice and rats, but was inactive in the MB and EPM despite reaching high concentrations in plasma. ADX71943 had no effect on BT, rotarod and sLMA. ADX71943 showed consistent and target-related efficacy in tests of disorders that have a significant peripheral component (acute and chronic pain), while having no effect in those associated with centrally-mediated anxiety-like reactivity and side effects. Thus, ADX71943 is a useful pharmacological tool for delineation of peripherally- versus centrally-mediated effects of GABA(B) receptor activation.

Allosteric Modulation of GABA-A Receptors by Different Drugs

Understanding how drugs allosterically modulate GABA-A receptor (GABAAR) channel function remains a challenge. Anesthetic drugs like etomidate, propofol and pentobarbital bind at transmembrane domain (TMD) inter-subunit interfaces at different locations. Benzodiazepines bind at the αγ interface in the extracellular domain (ECD) and neurosteroids are believed to bind within the α-subunit TMD helix bundle. A common feature of these drugs is that they potentiate GABA-induced currents. Here, we examined whether structural mechanisms underlying their functional effects are distinct. We monitored motions induced individually by these drugs at each inter-subunit TMD interface and also tested whether modulation caused by pairs of drugs binding to distinct sites are additive or super-additive. We individually introduced a cysteine in the M1 helices of each subunit at α1I227, β2L223 and γ2LI238 to probe the different inter-subunit interfaces. We expressed wild-type and mutant α1β2γ2L GABAARs in Xenopus oocytes and measured rates of modification of the substituted cysteines in the absence and presence of GABA and different allosteric modulators: the anesthetics pentobarbital (PB) and etomidate, the neurosteroid THDOC and the benzodiazepine flurazepam. GABA activation significantly increased MTSEA modification of αI227C, βL223C and γI238C. PB activation increased modification of αI227C and γI238C but not βL223C. Modulating concentrations of PB or etomidate had no effects. THDOC significantly increased the rate of modification of βL223C whereas flurazepam increased γI238C modification. We found that potentiation of EC10 GABA currents by combining modulating concentrations of THDOC and flurazepam was the sum of the potentiation produced by the two drugs individually. Combining PB and THDOC produced potentiation more than the sum of the potentiation produced by the two drugs individually. Our data suggests that drugs binding to different sites act via distinct mechanisms. Further analysis is underway to determine if these mechanisms are independent or coupled.

γ-Aminobutyric Acid (GABA) Imparts Partial Protection from Heat Stress Injury to Rice Seedlings by Improving Leaf Turgor and Upregulating Osmoprotectants and Antioxidants

Four-day-old rice (Oryza sativa L.) seedlings were subjected to varying temperatures of 30/20, 35/25, and 42/37 °C [light/dark (15/9 h); light intensity: 350 μmol m−2 s−1, RH 65–70 %] in glass Petri dishes for 10 days in the absence (control) or the presence of γ-aminobutyric acid (GABA) 1 mM under the controlled conditions of a growth chamber. With rise in temperature, the length of both shoots and roots was inhibited severely and there was a marked decrease in survival, especially at 42/37 °C. Endogenous GABA content increased more than twofold in moderately stressed (MS) 35/25 °C plants, whereas it decreased sevenfold in severely stressed (SS) 42/37 °C plants compared to MS plants, and this decrease was associated with marked reduction in growth and survival. Exogenous application of GABA to the heat-stressed plants significantly improved growth as well as survival. It was linked to reduction in damage to membranes, improvement in cellular reducing ability, chlorophyll content, and photochemical efficiency in shoots. Relative leaf water content and stomatal conductance were also improved with the application of GABA and their improvement was related to increased accumulation of the osmolytes proline and trehalose. In the presence of GABA, the shoots suffered less oxidative damage in terms of malondialdehyde and hydrogen peroxide contents. The activities of enzymatic antioxidants such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase were severely inhibited in plants growing at 42/37 °C compared to those growing at 35/25 °C. The nonenzymatic antioxidants like ascorbate and glutathione followed a similar pattern. GABA-treated SS plants showed enhanced levels of enzymatic and nonenzymatic antioxidants compared to untreated controls. Thus, GABA appears to impart partial protection from heat stress to rice plants by elevating leaf turgor due to increased accumulation of osmolytes and reduction of oxidative damage by stimulation of antioxidants. These findings provided evidence about the involvement of GABA in governing heat sensitivity in rice.

Pharmacological properties of AC-3933, a novel benzodiazepine receptor partial inverse agonist

We investigated in this study the pharmacological properties of AC-3933 (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-1,6-naphthyridin-2(1H)-one), a novel benzodiazepine receptor (BzR) partial inverse agonist. AC-3933 potently inhibited [3H]-flumazenil binding to rat whole brain membrane with a Ki value of 5.15±0.39nM and a GABA ratio of 0.84±0.03. AC-3933 exhibited almost no affinity for the other receptors, transporters and ion channels used in this study. In addition, AC-3933, in the presence of GABA (1μM), gradually but significantly increased [35S] tert-butylbicyclophosphorothionate binding to rat cortical membrane to 117.1% of the control (maximum increase ratio) at 3000nM. However, this increase reached a plateau at 30nM with hardly any change at a concentration range of 100–3000nM (from 115.2% to 117.1%). AC-3933 (0.1–10μM) significantly enhanced KCl-evoked acetylcholine (ACh) release from rat hippocampal slices in a concentration-dependent manner. Moreover, in vivo brain microdialysis showed that intragastric administration of AC-3933 at the dose of 10mg/kg significantly increased extracellular ACh levels in the hippocampus of freely moving rats (area under the curve (AUC0–2h) of ACh level; 288.3% of baseline). These results indicate that AC-3933, a potent and selective BzR inverse agonist with low intrinsic activity, might be useful in the treatment of cognitive disorders associated with degeneration of the cholinergic system.

State-Dependent Etomidate Binding in GABAA Receptors Probed with Cysteine Substitution and Protection from Modification

Background: A central tenet of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. This is a difficult concept to test experimentally, because in most agonist-receptor systems, the act of assessing agonist binding affinity results in receptor activation. In α1β2γ2L GABAA receptors, the anesthetic etomidate is an allosteric agonist at high concentrations, and potentiates GABA activation at low concentrations. Sites mediating both actions were photolabeled with azi-etomidate, identifying the β2M286 sidechain as a contact point. We used a cysteine substitution and sulfhydryl modification to study etomidate interactions with this sidechain. Methods: using two-microelectrode voltage clamp electrophysiology in Xenopus oocytes, we characterized etomidate agonism and potentiation of GABA activation in α1β2M286Cγ2L GABAA receptors. We studied covalent modification of the β2M286C sidechain by a sulfhydryl-selective reagent, para-chloromercuribenzenesulfonate (pCMBS) with and without GABA. Etomidate-dependent protection of the sulfhydryl was also assessed. Results: Oocyte-expressed α1β2M286Cγ2L receptors displayed reduced sensitivity to GABA, and no agonism by etomidate. However, etomidate still enhanced GABA-activated currents from α1β2M286Cγ2L receptors. Exposure of α1β2M286Cγ2L receptors to pCMBS irreversibly increased activation by low (EC10) GABA. The apparent rate of pCMBS modification increased with addition of GABA. Etomidate in a concentration-dependent manner reduced the rate of β2M286C modification by pCMBS. Etomidate protection at β2M286C was enhanced in the presence of GABA. Conclusions: Etomidate, like azi-etomidate, binds next to the β2M286 residue. Based on its apparent protectant potency, the affinity of etomidate for GABA-bound receptors is greater than that for resting receptors, as expected. Our results have important implications for both designing and interpreting experiments that use mutations to map allosteric modulator/agonist sites.Support: NIH (R01GM89745 and P01GM58448)

Chloride currents in cones modify feedback from horizontal cells to cones in goldfish retina

In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell (HC) interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in temporal and spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is achieved is unknown. Using electrophysiological techniques in the isolated retina of the goldfish, it was found that opening Ca(2+)-dependent Cl(-) channels in recorded cones reduced the size of feedback responses measured in both cones and HCs. Furthermore, we show that cones express Cl(-) channels that are gated by GABA released from HCs. Similar to activation of I(Cl(Ca)), opening of these GABA-gated Cl(-) channels reduced the size of light-induced feedback responses both in cones and HCs. Conversely, application of picrotoxin, a blocker of GABA(A) and GABA(C) receptors, had the opposite effect. In addition, reducing GABA release from HCs by blocking GABA transporters also led to an increase in the size of feedback. Because the independent manipulation of Ca(2+)-dependent Cl(-) currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl(-) current by itself is sufficient to reduce the size of HC feedback. However, additional effects of GABA on outer retinal processing cannot be excluded. These results can be accounted for by an ephaptic feedback model in which a cone Cl(-) current shunts the current flow in the synaptic cleft. The Ca(2+)-dependent Cl(-) current might be essential to set the initial balance between the feedforward and the feedback signals active in the cone HC synapse. It prevents that strong feedback from HCs to cones flood the cone with Ca(2)(+). Modulation of the feedback strength by GABA might play a role during light/dark adaptation, adjusting the amount of negative feedback to the signal to noise ratio of the cone output.

Gating-induced Conformational Rearrangement of the γ-Aminobutyric Acid Type A Receptor β-α Subunit Interface in the Membrane-spanning Domain

GABA(A) receptors mediate fast inhibitory synaptic transmission. The transmembrane ion channel is lined by a ring of five α helices, M2 segments, one from each subunit. An outer ring of helices comprising the alternating M1, M3, and M4 segments from each subunit surrounds the inner ring and forms the interface with the lipid bilayer. The structural rearrangements that follow agonist binding and culminate in opening of the ion pore remain incompletely characterized. Propofol and other intravenous general anesthetics bind at the βM3-αM1 subunit interface. We sought to determine whether this region undergoes conformational changes during GABA activation. We measured the reaction rate of p-chloromercuribenzenesulfonate (pCMBS) with cysteines substituted in the GABA(A) receptor α1M1 and β2M3 segments. In the presence of GABA, the pCMBS reaction rate increased significantly in a cluster of residues in the extracellular third of the α1M1 segment facing the β2M3 segment. Mutation of the β2M2 segment 19' position, R269Q, altered the pCMBS reaction rate with several α1M1 Cys, some only in the resting state and others only in the GABA-activated state. Thus, β2R269 is charged in both states. GABA activation induced disulfide bond formation between β2R269C and α1I228C. The experiments demonstrate that α1M1 moves in relationship to β2M2R269 during gating. Thus, channel gating does not involve rigid body movements of the entire transmembrane domain. Channel gating causes changes in the relative position of transmembrane segments both within a single subunit and relative to the neighboring subunits.

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABAA receptors

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB(1) antagonists / inverse agonists. Concentrations of 0.5-10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA(A) receptors but found a significant positive allosteric modulation instead. Recombinant GABA(A) receptors were expressed in Xenopus oocytes. Receptors were exposed to AM251 or rimonabant in the absence and presence of GABA. Standard electrophysiological techniques were used to monitor the elicited ionic currents. AM251 dose-dependently potentiated responses to 0.5 µM GABA at the recombinant α(1) β(2) γ(2) GABA(A) receptor with an EC(50) below 1 µM and a maximal potentiation of about eightfold. The Hill coefficient indicated that more than one binding site for AM251 was located in this receptor. Rimonabant had a lower affinity, but a fourfold higher efficacy. AM251 potentiated also currents mediated by α(1) β(2) , α(x) β(2) γ(2) (x = 2,3,5,6), α(1) β(3) γ(2) and α(4) β(2) δ GABA(A) receptors, but not those mediated by α(1) β(1) γ(2) . Interestingly, the CB(1) receptor antagonists LY320135 and O-2050 did not significantly affect α(1) β(2) γ(2) GABA(A) receptor-mediated currents at concentrations of 1 µM. This study identified rimonabant and AM251 as positive allosteric modulators of GABA(A) receptors. Thus, potential GABAergic effects of commonly used concentrations of these compounds should be considered in in vitro experiments, especially at extrasynaptic sites where GABA concentrations are low. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Influences on blockade by t‐butylbicyclo‐phosphoro‐thionate of GABAA receptor spontaneous gating, agonist activation and desensitization

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1β2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1β2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)β2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1β2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)β2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1β2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1β2γ2 receptors seen with GABA (1 μm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)β2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.

Efficacy of Synaptic Inhibition Depends on Multiple, Dynamically Interacting Mechanisms Implicated in Chloride Homeostasis

Chloride homeostasis is a critical determinant of the strength and robustness of inhibition mediated by GABAA receptors (GABAARs). The impact of changes in steady state Cl− gradient is relatively straightforward to understand, but how dynamic interplay between Cl− influx, diffusion, extrusion and interaction with other ion species affects synaptic signaling remains uncertain. Here we used electrodiffusion modeling to investigate the nonlinear interactions between these processes. Results demonstrate that diffusion is crucial for redistributing intracellular Cl− load on a fast time scale, whereas Cl−extrusion controls steady state levels. Interaction between diffusion and extrusion can result in a somato-dendritic Cl− gradient even when KCC2 is distributed uniformly across the cell. Reducing KCC2 activity led to decreased efficacy of GABAAR-mediated inhibition, but increasing GABAAR input failed to fully compensate for this form of disinhibition because of activity-dependent accumulation of Cl−. Furthermore, if spiking persisted despite the presence of GABAAR input, Cl− accumulation became accelerated because of the large Cl− driving force that occurs during spikes. The resulting positive feedback loop caused catastrophic failure of inhibition. Simulations also revealed other feedback loops, such as competition between Cl− and pH regulation. Several model predictions were tested and confirmed by [Cl−]i imaging experiments. Our study has thus uncovered how Cl− regulation depends on a multiplicity of dynamically interacting mechanisms. Furthermore, the model revealed that enhancing KCC2 activity beyond normal levels did not negatively impact firing frequency or cause overt extracellular K− accumulation, demonstrating that enhancing KCC2 activity is a valid strategy for therapeutic intervention.

Ginseng derivative ocotillol enhances neuronal activity through increased glutamate release: a possible mechanism underlying increased spontaneous locomotor activity of mice

Ginsenosides are the main active ingredients in ginseng and have recently been reported to have beneficial effects on the CNS. Ocotillol is a derivate of pseudoginsenoside-F11, which is an ocotillol-type ginsenoside found in American ginseng. We examined the effects of ocotillol (a) on neuronal activity of projection neurons, mitral cells (MC), in a mouse olfactory bulb brain slice preparation using whole-cell patch-clamp recording, and (b) on animal behavior by measuring locomotor activity of mice in vivo. Ocotillol displayed an excitatory effect on spontaneous action potential firing and depolarized the membrane potential of MCs. The effect was concentration-dependent, with an EC(50) of 4 μM. In the presence of blockers of ionotropic glutamatergic and GABAergic synaptic transmission (6-cyano-7-nitroquinoxaline-2,3-dione [CNQX], 10 μM; D-AP5, 50 μM; gabazine, 5 μM), the excitatory effect of ocotillol on firing was abolished. Further experiments showed that the ocotillol-induced neuronal excitation persisted in the presence of GABA(A) receptor antagonist gabazine but was eliminated by applying AMPA/kainate receptor antagonist CNQX and N-methyl-d-aspartate (NMDA) receptor antagonist D-AP5, suggesting that ionotropic glutamate transmission was involved in mediating the effects of ocotillol. Bath application of ocotillol evoked an inward current as well as an increased frequency of spontaneous glutamatergic excitatory postsynaptic currents (EPSCs). Both the inward current and sEPSCs could be blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. These results indicate that the excitatory action of ocotillol on MCs was mediated by enhanced glutamate release. Behavioral experiments demonstrated that ocotillol increased locomotor activities of mice. Our results suggest that ocotillol-evoked neuronal excitability was mediated by increased release of glutamate, which may be responsible for the increased spontaneous locomotor activities in vivo.

Oral GABA treatment downregulates inflammatory responses in a mouse model of rheumatoid arthritis

Current treatments for rheumatoid arthritis (RA) have long-term side effects such that new treatments are needed that can safely help manage the disease. There is a growing appreciation that GABA receptors (GABA-Rs) on immune cells provide new targets that can be used to modulate immune cell activity. Here, we show for the first time that activation of peripheral GABA-Rs can inhibit the development of disease in the collagen-induced arthritis (CIA) mouse model of RA. Mice that received oral GABA had a reduced incidence of CIA, and those mice that did develop CIA had milder symptoms. T cells from GABA-treated mice displayed reduced proliferative responses to collagen and their APC had a reduced ability to promote the proliferation of collagen-reactive T cells. Thus, GABA downregulated both T-cell autoimmunity and APC activity. Collagen-reactive T cells from GABA-treated mice displayed reduced recall responses in the presence of GABA ex vivo, indicating that GABA consumption did not desensitize these cells to GABA. GABA-treated mice had reduced collagen-reactive IgG2a, but not IgG1 antibodies, consistent with reduced Th1 help. The levels of serum anti-collagen IgG2a antibodies were correlated significantly with the CIA disease scores of individual mice. Our results suggest that activation of peripheral GABA-Rs may provide a new modality to modulate T cell, B cell, and APC activity and help ameliorate RA and other inflammatory diseases.

Perinatal exposure to environmental polychlorinated biphenyls sensitizes hippocampus to excitotoxicity ex vivo

Ortho-substituted polychlorinated biphenyls (PCBs) are a concern to human developmental health. Rat dams were exposed to an environmentally relevant mixture of PCBs, Aroclor 1254, or pure congener PCB 95 (6 mg/kg/day) during the perinatal period (GD 5 through PD 21). Hippocampal slices prepared from offspring 1-3 weeks post-weaning were tested for persisting changes in sensitivity to the excitotoxicant picrotoxin. Hippocampal slices were placed on multielectrode arrays. Field excitatory postsynaptic potentials (fEPSPs) were recorded from Schaffer Collateral/Commissural fibers in striatum radiatum of the CA1 region in response to single pulse stimuli. After recording baseline excitability, GABA(A) receptors were blocked by inclusion of picrotoxin (100 μM) in the aCSF perfusate. Picrotoxin produced negligible changes in fEPSP slope in slices isolated from offspring exposed to vehicle, whereas slices from either PCB test group invariably showed >200% (p<0.01) synaptic facilitation. Picrotoxin produced prominent after-discharges (epileptic wave forms) in the evoked potentials measured from PCB exposed, but not control, hippocampal slices. These results show that developmental exposure to non-coplanar PCBs is sufficient to produce changes in synaptic plasticity that can be unmasked in the presence of GABA(A) receptor deficits that persist 1-3 weeks after exposure ceased. Developmental exposure to PCBs may sensitize seizure susceptibility postnatally, especially in susceptible populations with GABA(A) receptor signaling deficits.