GABA-gated Cl Research Articles

GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), β(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2β2γ2-GABAARs versus extrasynaptic type α4β3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2β2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2β2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2β2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits

Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate inhibitory transmission in the central nervous system. Cell surface expression of GABAARs, a prerequisite for their function, is dependent on the appropriate assembly of the receptor subunits and their transient interactions with molecular chaperones within the endoplasmic reticulum (ER) and Golgi apparatus. Here, we describe a highly conserved amino acid sequence within the extracellular N-terminal domain of the receptor subunits adjoining the first transmembrane domain as a region important for GABAAR processing within the ER. Modifications of this region in the α1, β3, and γ2 subunits using insertion or site-directed mutagenesis impaired GABAAR trafficking to the cell surface in heterologous cell systems although they had no effect on the subunit assembly. We found that mutated receptors accumulated in the ER where they were shown to associate with chaperones calnexin, BiP, and Grp94. However, their surface expression was increased when ER-associated degradation or proteosome function was inhibited, while modulation of ER calcium stores had little effect. When compared to the wt, mutated receptors showed decreased interaction with calnexin, similar binding to BiP, and increased association with Grp94. Structural modeling of calnexin interaction with the wt or mutated GABAAR revealed that disruption in structure caused by mutations in the conserved region adjoining the first transmembrane domain may impair calnexin binding. Thus, this previously uncharacterized region plays an important role in intracellular processing of GABAARs at least in part by stabilizing their interaction with calnexin.

Broflanilide, a Meta-Diamide Insecticide Seed Treatment for Protection of Wheat and Mortality of Wireworms (Agriotes obscurus) in the Field.

Following the deregistration of lindane, several neonicotinoid insecticides have been registered as seed treatments for controlling wireworms in cereal crops. Unlike lindane, which did both, neonicotinoids provide crop protection but do not reduce wireworm populations. Hence populations of various economic species are growing in key wheat production areas of the United States and Canada, and there is a need for novel seed treatments that match the efficacy of lindane. Herein we evaluated broflanilide, a novel GABA-gated Cl- channel allosteric modulator that differs from the cyclodienes (e.g., lindane) in that it binds to a unique site in the GABA receptor. As such, broflanilide confers a new mode of insecticidal action (Group 30 MOA), and is the first meta-diamide insecticide developed. When evaluated in field trials over 7 yr at various rates, broflanilide at 5.0 g [AI]/100 kg wheat seed was as effective at protecting wheat stand from wireworm (Agriotes obscurus) injury as the current industry standard thiamethoxam at 20-30 g [AI]/100 kg seed. In addition, broflanilide at 5.0 g reduced neonate wireworms (produced from eggs laid in established wheat during the growing season) and resident wireworms (in the field at time of planting) by 73.1 and 81.1%, respectively, which is comparable to that reported for the previous industry standard lindane (75.3 and 57.6%, respectively). These studies show that broflanilide at 5.0 g [AI] will provide consistent wheat stand protection (equal to thiamethoxam at 20-30 g [AI]), and A. obscurus wireworm population reduction (equal to lindane at 59 g [AI]), and will do so at far lower dosages per hectare.

Functional Coupling of K+-Cl- Cotransporter (KCC) to GABA-Gated Cl- Channels in the Central Nervous System of Drosophila melanogaster Leads to Altered Drug Sensitivities.

GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, we employed electrophysiological, genetic, and pharmacological methods to characterize the physiological underpinnings of KCC function to the Drosophila CNS. Our data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly ( P < 0.05) reduces the CNS sensitivity to γ-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant ( P < 0.001) increase in CNS spontaneous activity over baseline firing rates that supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides, as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease.

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl− from acidic organelles in amacrine cells

γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl- However, the electrochemical gradient for Cl- across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl- levels support inhibition, whereas higher levels of cytosolic Cl- can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl- from acidic organelles and transiently elevates cytosolic Cl-, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl- We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl- channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl- To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl- from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl- from acidic organelles.NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl- from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl- levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.

Combined diazepam and MK-801 therapy provides synergistic protection from tetramethylenedisulfotetramine-induced tonic–clonic seizures and lethality in mice

The synthetic rodenticide, tetramethylenedisulfotetramine (TMDT), is a persistent and highly lethal GABA-gated Cl− channel blocker. TMDT is clandestinely produced, remains popular in mainland China, and causes numerous unintentional and deliberate poisonings worldwide. TMDT is odorless, tasteless, and easy to manufacture, features that make it a potential weapon of terrorism. There is no effective treatment. We previously characterized the effects of TMDT in C57BL/6 mice and surveyed efficacies of GABAergic and glutamatergic anticonvulsant treatments. At 0.4mg/kg i.p., TMDT produced neurotoxic symptomatology consisting of twitches, clonic and tonic–clonic seizures, often progressing to status epilepticus and death. If administered immediately after the occurrence of the first clonic seizure, the benzodiazepine diazepam (DZP) effectively prevented all subsequent seizure symptoms, whereas the NMDA receptor antagonist dizocilpine (MK-801) primarily prevented tonic–clonic seizures. The latter agent, however, appeared to be more effective at preventing delayed death. The present study further explored these phenomena, and characterized the therapeutic actions of DZP and MK-801 as combinations. Joint treatment with both DZP and MK-801 displayed synergistic protection against tonic–clonic seizures and 24h lethality as determined by isobolographic analysis. Clonic seizures, however, remained poorly controlled. A modification of the treatment regimen, where DZP was followed 10min later by MK-801, yielded a reduction in both types of seizures and improved overall outcome. Simultaneous monitoring of subjects via EEG and videography confirmed effectiveness of this sequential regimen. We conclude that TMDT blockage at GABAA receptors involves early activation of NMDA receptors, which contribute to persistent ictogenic activity. Our data predict that a sequential combination treatment with DZP followed by MK-801 will be superior to either individual therapy with, or simultaneous administration of, these two agents in treating TMDT poisoning.

BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor, Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein-protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl(-) influx through the regulation of cell surface expression of GABAARs.

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.

Two types of chloride transporters are required for GABAAreceptor-mediated inhibition inC. elegans

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

Chloride Accumulation Drives Volume Dynamics Underlying Cell Proliferation and Migration

During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cell's ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl- along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl- to approximately 100 mM, a concentration threefold greater than predicted by the Nernst equation. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl- channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl- as indicated by a 40-mM decrease in [Cl-]i. These findings provide a new framework for considering the role of intracellular Cl- in glioma cells. Here, Cl- serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well.

Molecule of the Month

A new, non-SSRI mechanism of action for Prozac™. Unquestionably, Prozac™ was a landmark drug that redefined the treatment for depression, anxiety disorders, premenstrual syndrome and post-traumatic stress disorder as well as ushering in a wave of new therapies targeting specific serotonin (5-HT) reuptake inhibition (SSRI) [1]. In a recent manuscript (Psychopharmacology 2006, 186, 362-372), Pinna and co-workers report that fluoxetine and its N-desmethyl congener norfluoxetine stereospecifically and selectively increase brain neurosteroid content at doses that are inactive on 5-HT reuptake [2]. Pinna further states that the term ‘SSRI’ may be misleading in describing the pharmacological profile of fluoxetine and its congeners and suggests the term ‘selective brain steroidogenic stimulants’ (SBSSs) as a better descriptor [2]. Recently, it has been shown that neurosteroids, such as allopregnanolone (Allo), play critical roles in human brain pathophysiology such as anxiety disorders, premenstrual syndrome, posttraumatic stress disorder, postpartum depression and general depression. For the treatment of major depression, recent clinical trials have indicated that the pharmacological profiles of Prozac™ and related congeners correlate with an increase of Allo in the brain and cerebrospinal fluid. Importantly, Allo is a potent (nM affinity) positive allosteric modulator of gamma-aminobutyric acid (GABA) action at GABAA receptors, potentiating the intensity of GABAgated Cl- currents [3]. Thus, Prozac™ and related analogs increase Allo levels, and subsequent activation of GABAA receptors, at currently prescribed clinical doses which may not block 5-HT reuptake, but does effectively treat depression, premenstrual dysphoria and anxiety [2]. Pinna then explored this hypothesis in animal behavioral models of aggression and anxiety [2]. Their studies found that social isolation in mice is correlated with a down-regulation of Allo content and that pretreatment with Allo abolished aggressiveness as well as correcting the altered responses of GABAA receptors to specific agonists (pentobarbital) and antagonists (picrotoxin). Therefore, drugs capable of upregulating brain Allo content normalize behavioral abnormalities expressed in mice with a downregulation of brain Allo levels. Significantly, Pinna and co-workers discovered that the actions of fluoxetine and norfluoxetine on brain Allo content are stereoselective whereas 5-HT reuptake lacks stereospecificity. Prozac™ is a racemic compound. Pinna found that the EC50s of the (S)-enantiomers of fluoxetine and norfluoxetine to normalize brain Allo levels are 10- and 50-fold lower than those required to inhibit 5-HT reuptake [2]. Will this research herald new drug discovery campaigns aimed at identifying compounds capable of selectively increasing brain Allo levels under the moniker SBSSs? REFERENCES [1] Wong, D.T.; Bymaster, F.P.; Reid, L.R.; Mayle, D.A.; Krushinski, J.H.; Robertson, D.W. Norfluoxetine enetiomers as inhibitors of serotonin uptake in rat brain. Neuropsychopharmacology 1993, 8, 337-344. [2] Pinna, G.; Costa, E.; Guidotti, A. Fluoxetine and norfluoxetine stereospecifically and selectively increase brain neurosteroid content at doses that are inactive on 5-HT reuptake. Psychopharmaology 2006, 186, 362-372. [3] Lambert, J.J.; Belelli, D.; Peden, D.R.; Vardy, A.W.; Peters, J.A. Neurosteroid modualtion of GABBA receptors. Prog. Neurobiol. 2003, 71, 67-80.

The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.

The regulation of α-MSH release by GABA is mediated by a chloride-dependent [Ca 2+] c increase in frog melanotrope cells

In frog melanotrope cells, γ-aminobutyric acid (GABA) induces a biphasic effect, i.e. a transient stimulation followed by a more sustained inhibition of α-MSH release, and both phases of the GABA effect are mediated by GABA A receptors. We have previously shown that the stimulatory phase evoked by GABA A receptor agonists can be accounted for by calcium entry. In the present study, we have investigated the involvement of the chloride flux on GABA-induced [Ca 2+] c increase and α-MSH release. We show that GABA evokes a concentration-dependent [Ca 2+] c rise through specific activation of the GABA A receptor. The GABA-induced [Ca 2+] c increase results from opening of voltage-activated L- and N-type calcium channels, and sodium channels. Variations of the extracellular Cl − concentration revealed that GABA-induced [Ca 2+] c rise and α-MSH release both depend on the Cl − flux direction and driving force. These observations suggest for the first time that GABA-gated Cl − efflux provokes an increase in [Ca 2+] c increase that is responsible for hormone secretion.

Flavonoid modulation of ionic currents mediated by GABA A and GABA C receptors

The modulation of ionotropic γ-aminobutyric acid (GABA) receptors (GABA-gated Cl − channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by α 1β 1γ 2s GABA A and ρ 1 GABA C receptors expressed in Xenopus laevis oocytes in the micromolar range. α 1β 1γ 2s GABA A and ρ 1 GABA C receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by α 4β 2 neuronal nicotinic acetylcholine, serotonin 5-HT 3A and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized α 1β 1γ 2s GABA A receptors. Effects of apigenin and quercetin on α 1β 1γ 2s GABA A receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.

On the putative physiological role of allopregnanolone on GABA A receptor function

To obtain definitive evidence for a physiological allosteric modulatory role for endogenous brain ALLO on GABA A receptor function, we studied GABA A receptor activity under conditions in which the concentration of endogenous brain ALLO was decreased by about 80% for longer than 5 h following the administration of SKF 105111- 17β-17-[bis (1methylethyl) amino carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF), a potent inhibitor of 5α-reductases Type I and II. We used the in situ patch–clamp technique to record GABA-evoked currents and spontaneous inhibitory postsynaptic currents (sIPSCs) from pyramidal neurons in neocortical slices of vehicle- or SKF-treated mice. The potency, but not the efficacy, of exogenously applied GABA was decreased in slices from mice treated with SKF. When neocortical slices were treated in vitro for 3 h with 10μM SKF, ALLO was also reduced (25–30%) and in addition, the GABA dose–response curve was shifted to the right; however this shift was not as marked as the shift in the slices obtained from mice treated with SKF, in keeping with the smaller decrease of the ALLO content in these slices. Furthermore, direct application of ALLO to these slices shifted the dose–response curve of GABA back toward a non-SKF treated profile. We then analyzed GABAergic sIPSCs in neocortical slices obtained from vehicle or SKF-treated mice. Mean decay time and charge transfer were significantly reduced by SKF treatment. The decay of sIPSCs was best fitted by two exponentials, but only the fast component was decreased in the SKF group. Direct application of ALLO (100nM) normalizes the sIPSC kinetics in slices from ALLO depleted mice. No changes were detected in the amplitude or frequency of sIPSCs. These data demonstrate that endogenous ALLO physiologically regulates spontaneously induced Cl − current by acting on a specific recognition site, which is probably located on GABA A receptors (a receptor on a receptor), thereby prolonging inhibitory currents by facilitating conformational transition of the GABA-gated Cl − channel to an open state.

Control of intracellular chloride concentration and GABA response polarity in rat retinal ON bipolar cells.

GABAergic modulation of retinal bipolar cells plays a crucial role in early visual processing. It helps to form centre-surround receptive fields which filter the visual signal spatially at the bipolar cell dendrites in the outer retina, and it produces temporal filtering at the bipolar cell synaptic terminals in the inner retina. The observed chloride transporter distribution in ON bipolar cells has been predicted to produce an intracellular chloride concentration, [Cl(-)](i), that is significantly higher in the dendrites than in the synaptic terminals. This would allow dendritic GABA-gated Cl(-) channels to generate the depolarization needed for forming the lateral inhibitory surround of the cell's receptive field, while synaptic terminal GABA-gated Cl(-) channels generate the hyperpolarization needed for temporal shaping of the light response. In contrast to this idea, we show here that in ON bipolar cells [Cl(-)](i) is only slightly higher in the dendrites than in the synaptic terminals, and that GABA-gated channels in the dendrites may generate a hyperpolarization rather than a depolarization. We also show that [Cl(-)](i) is controlled by movement of Cl(-) through ion channels in addition to transporters, that changes of [K(+)](o) alter [Cl(-)](i) and that voltage-dependent equilibration of [Cl(-)](i) in bipolar cells will produce a time-dependent adaptation of GABAergic modulation with a time constant of 8 s after illumination-evoked changes of membrane potential. Time-dependent adaptation of [Cl(-)](i) to voltage changes in retinal bipolar cells may add a previously unsuspected layer of temporal processing to signals as they pass through the retina.

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species.

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.

Exploring the structural basis of neurotoxicity in C(17)-polyacetylenes isolated from water hemlock.

Water hemlock, Cicuta virosa, belonging to the Umbelliferae, is well-known as a toxic plant responsible for lethal poisonings in humans as well as animals, causing tonic and clonic convulsions and respiratory paralysis. Cicutoxin (1), being a major violent toxin of the plant, is a chemical in the class of C(17)-polyacetylenes bearing a long pi-bond conjugation system, a terminal hydroxyl, and an allylic hydroxyl in its structure, and a variety of its analogues have been isolated from the plant. In the present study, various derivatives of these toxins were synthesized through acetylation, methylation, and oxidation of cicutoxin (1) and virol A (3) and B (4). 1-Dehydroxyvirol A (28) was prepared through the coupling of (7S)-dodeca-3,5-dien-1-yn-7-ol and 1-iodopentyne under Sonogashira's conditions. A monoacetylenic compound (29) was also prepared through the coupling of (5S)-1-chlorodeca-1,3-dien-5-ol and 1-iodopentyn-5-ol. The structure-activity relationships involved in the acute toxicity of cicutoxin derivatives in mice were investigated, and the length and geometry of pi-bond conjugation and the O-functional groups were found to be important for activity. The potency in inhibition of the specific binding of the noncompetitive GABA antagonist, [(3)H]EBOB, to GABA-gated Cl(-) channels of GABA receptors in rat brain cortex was found to be correlated with acute toxicity, indicating that the ability to bind to these channels plays an important role in the acute toxicity of these compounds.

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes.

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.

Brain allopregnanolone regulates the potency of the GABA A receptor agonist muscimol

Allopregnanolone (ALLO), a potent positive-allosteric modulator of the action of GABA at GABA A receptors, is synthesized in the brain from progesterone by the sequential action of two enzymes: 5α-reductase and 3 α-hydroxysteroidoxidoreductase. The concentration of ALLO in various parts of the mouse brain varies substantially, from 15 pmol/g in the olfactory bulb, to approximately 6 pmol/g in the frontoparietal cortex, and 2.7 pmol/g in the cerebellum. The systemic administration of 48 μmol/kg of the Type I and Type II 5α-reductase inhibitor, (17β)-17-[bis (1-methylethyl) amino carbonyl)] androsta-3, 5-diene-3-carboxylic acid (SKF 105,111), reduced brain ALLO content by 80–90% in 30 min; the rate constant ( k) of ALLO decrease in each brain area can be utilized to establish the rate of ALLO biosynthesis, which is higher in the olfactory bulb (62 pmol/g/h) than in the frontoparietal cortex (24 pmol/g/h) or cerebellum (11 pmol/g/h). The duration of the righting reflex loss elicited by the potent GABA A receptor agonist muscimol was reduced in SKF 105,111-treated ALLO-depleted mice. SKF 105,111 treatment had no effect on muscimol metabolism or on brain levels of pregnenolone and progesterone; however, the brain levels of 5α-DHP, the precursor of ALLO, were also decreased. Administration of ALLO at a dose of 15 μmol/kg ip by itself did not alter the muscimol-induced loss of the righting reflex; but it completely blocked the effect of SKF 105,111. To elucidate the possible molecular mechanism by which a decrease of brain ALLO content can shorten the duration of the righting reflex loss elicited by muscimol, we patch-clamped neocortical pyramidal neurons of mice pretreated with SKF 105,111 or vehicle, and studied the efficiency of muscimol in eliciting Cl − currents. The current amplitude was significantly smaller in neurons from SKF 105,111-treated mice, especially at lower doses (0.1–1 μM) of muscimol, and the muscimol dose–response (0.1–10 μM) relationship displayed cooperativity ( n H=1.4). These data suggest that ALLO synthesized in brain plays an important physiological permissive role in the modulation of GABA-gated Cl − channel function.