Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.     

Bisphenol a inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.     

Laccase treatment impairs bisphenol A-induced cancer cell proliferation affecting estrogen receptor alpha-dependent rapid signals

A wide variety of environmental contaminants exert estrogenic actions in wildlife, laboratory animals, and in human beings through binding to nuclear estrogen receptors (ERs). Here, the mechanism(s) of bisphenol A (BPA) to induce cell proliferation and the occurrence of its bioremediation by treatment with laccase are reported. BPA, highly present in natural world and considered as a model of environmental estrogen action complexity, promotes human cancer cell proliferation via ERalpha-dependent signal transduction pathways. Similar to 17beta-estradiol, BPA increases the phosphorylation of both extracellular regulated kinase and AKT. Specific inhibitors of these kinase completely block the BPA effect on cancer cell proliferation. Notably, high BPA concentrations (i.e., 0.1 and 1 mM) are cytotoxic even in ERalpha-devoid cancer cells, indicating that an ERalpha-independent mechanism participates to BPA-induced cytotoxicity. On the other hand, BPA oxidation by laccase impairs the binding of this environmental estrogen to ERalpha loosing at all ERalpha-dependent effect on cancer cell proliferation. Moreover, the laccase-catalyzed oxidation of BPA reduces the BPA cytotoxic effect. Thus, laccase appears to impair BPA action(s), representing an invaluable bioremediation enzyme.     

Bisphenol A exposure in utero disrupts early oogenesis in the mouse

Estrogen plays an essential role in the growth and maturation of the mammalian oocyte, and recent studies suggest that it also influences follicle formation in the neonatal ovary. In the course of studies designed to assess the effect of the estrogenic chemical bisphenol A (BPA) on mammalian oogenesis, we uncovered an estrogenic effect at an even earlier stage of oocyte development—at the onset of meiosis in the fetal ovary. Pregnant mice were treated with low, environmentally relevant doses of BPA during mid-gestation to assess the effect of BPA on the developing ovary. Oocytes from exposed female fetuses displayed gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. In the mature female, these aberrations were translated into an increase in aneuploid eggs and embryos. Surprisingly, we observed the same constellation of meiotic defects in fetal ovaries of mice homozygous for a targeted disruption of ERβ, one of the two known estrogen receptors. This, coupled with the finding that BPA exposure elicited no additional effects in ERβ null females, suggests that BPA exerts its effect on the early oocyte by interfering with the actions of ERβ. Together, our results show that BPA can influence early meiotic events and, importantly, indicate that the oocyte itself may be directly responsive to estrogen during early oogenesis. This raises concern that brief exposures during fetal development to substances that mimic or antagonize the effects of estrogen may adversely influence oocyte development in the exposed female fetus.     

The environmental estrogenic compound bisphenol A exerts estrogenic effects on mouse hippocampal (HT-22) cells: neuroprotection against glutamate and amyloid beta protein toxicity

We have examined using immortalized clonal mouse hippocampal cell line (HT-22) whether the environmental estrogenic compound bisphenol A (BPA), like estrogen, has any neuroprotective effect against glutamate and amyloid beta protein-induced neurotoxicity. BPA protects HT-cells against both 5 mM glutamate and 2 microM amyloid beta protein-induced cell death in a dose dependent manner. Optimum protection was attained at 1 microM and 500 nM BPA against 5 mM glutamate and 2 microM amyloid beta protein-induced HT-22 cell death, respectively. Using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptors (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 1 microM BPA treatment for 24 h, followed by 20-h treatment with 5 mM glutamate, resulted in dramatic reduction in GR nuclear localization. We conclude that: (i) BPA mimics estrogen and exerts neuroprotective effects against both neurotoxins used; (ii) BPA inhibits enhanced nuclear localization of GR induced by glutamate; and (iii) HT-22 cells provide a good in vitro model system for screening the potencies of various environmental compounds for their estrogenic activity.     

4-Hydroxytamoxifen-induced cytotoxicity and bisphenol a: Competition for estrogen receptors in human breast cancer cell lines

Summary Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess potentially estrogenic effects of test compounds. As a result, in vitro screening methods such as cell proliferation assays using the estrogen-responsive human breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xenoestrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-induced cytotoxicity. This method measures the ability of various estrogenic compounds to compete with a known estrogen-receptor-mediated antihormonal drug, 4-hydroxytamoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamoxifen treatment of cells results in a dramatic decrease in mitochondrial dehydrogenase activity which is directly related to their estrogen-receptor content, inhibition of this effect with estrogenic compounds represents an estrogen-receptor interaction, or estrogenic rescue. The estrogenic compounds tested include a weak xenoestrogen, bisphernol A (BPA), and two biological estrogens, 17α- and 17β-estradiol. Competitive inhibition of 4-hydroxytamoxifen-induced cytotoxicity by BPA was compared to that of the biological estrogens. The results indicate that the biological estrogens can successfully compete with the antiestrogen in a dose-dependent manner. In addition, the assay is sensitive enough to detect estrogenic rescue by even the very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simple in vitro method could be used as an alternative or second-line screen for potential xenoestrogens.     

Effects of concanavalin A on desensitization kinetics of GABA responses inAchatina fulica neurons

The effects of the lectin concanavalin A (Con A), on the kinetics of desensitization of the responses of voltage clampedAchatina fulica LP5 neuron to microperfused acetylcholine (ACh) and GABA were compared. Both ACh and GABA elicited increases in chloride conductance which decayed biphasically during prolonged applications of these agonists; an initial rapid decay was followed by a later slow decay. Con A (5 g/ml) accelerated both the fast and the slow decays of responses to ACh. Con A (5 g/ml) also accelerated the fast decay of responses to GABA, but the slow decay was unaffected, even by 20 g/ml or more of the lectin. It is suggested that, at least in the case of GABA receptor, the fast and slow decays involve distinct desensitization kinetics. The effects of Con A on the desensitization of the ACh and GABA responses were reversed byd-mannose, a competitive and specific inhibitor of Con A binding to membrane sugar residues. These results provide further evidence that receptor desensitization can be influenced by perturbing the sugar moieties associated with the subunits comprising these signalling macromolecules. The carbohydrate residues may play an important role in regulating desensitization of transmitter receptors.Abbreviations ACh acetylcholine - Con A concanavalin A     

A method for the detection and characterization of GABA(A) receptors by calcium-sensitive fluorescent probes

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.     

Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture

We investigated effects of different concentrations (10(-7) - 10(-5) M) of bisphenol A (BPA), which is known as an estrogenic and anti-thyroid hormonal endocrine disrupter, on the expression of thyroid hormone receptor (TR) alpha and beta and retinoid X receptor (RXR) gamma mRNA in tails of stage 52-54 Xenopus tadpoles in organ culture in the presence or absence of different concentrations of triiodo-thyronine (T(3)). In the absence of T(3), BPA at any concentration examined did not show remarkable effects on tail length but blocked 10(-7) M T(3)-induced tail resorption in a concentration-dependent manner. Semi-quantitative analyses of TRalpha and TRbeta mRNAs by RT-PCR in the tail specimens indicated that BPA shows an apparent antagonistic effect towards the receptors and reduced their mRNA levels relative to controls. When administered together with 10(-7) M T(3), the antagonistic effects of BPA were detected more clearly and dose-dependently. While BPA prevented the autoinduction of both TRalpha and TRbeta genes by T(3), the effect was less marked on TRalpha than on TRbeta. BPA also moderately suppressed RXRgamma gene expression. Gene expression of RXRgamma, a partner for heterodimer formation of TRs, was supressed by T(3) alone and also by BPA alone, but no additive effects were observed so far as studied. The present study indicates that a relatively low concentration of BPA, 10(-7) M, as compared with those examined previously (10(-5) to 10(-4) M) by us and other investigators, acts as an antagonist of T(3) through suppression of TRalpha and TRbeta gene expression in Xenopus tail in culture.     

GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca(2+) in regulating the cell cycle.     

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glycinergic transmission shaped by the corelease of GABA in a mammalian auditory synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.     

Sleep is differently modulated by basal forebrain GABA(A) and GABA(B) receptors

There is evidence that GABA plays a major role in sleep regulation. GABA(A) receptor agonists and different compounds interacting with the GABA(A) receptor complex, such as barbiturates and benzodiazepines, can interfere with the sleep/wake cycle. On the other hand, there is very little information about the possible role of GABA(B) receptors in sleep modulation. The nucleus basalis of Meynert (NBM), a cholinergic area in the basal forebrain, plays a pivotal role in the modulation of sleep and wakefulness, and both GABA(A) and GABA(B) receptors have been described within the NBM. This study used unilateral infusions in the NBM to determine the effects of 3-hydroxy-5-aminomethylisoxazole hydrobromide (muscimol hydrobromide, a GABA(A) receptor subtype agonist) and beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen, a GABA(B) receptor subtype agonist) on sleep parameters in freely moving rats by means of polygraphic recordings. Muscimol (0.5 nmol) and baclofen (0.7 nmol) induced an increase in slow-wave sleep and an inhibition of wakefulness. Muscimol, but not baclofen, also caused a decrease in desynchronized sleep parameters. The results reported here indicate that 1) the NBM activation of both GABA(A) and GABA(B) receptors influences the sleep/wake cycle, and 2) GABA(A) but not GABA(B) receptors are important for desynchronized sleep modulation, suggesting that the two GABAergic receptors play different roles in sleep modulation.     

Prenatal exposure to low doses of bisphenol A alters the periductal stroma and glandular cell function in the rat ventral prostate

Environmental estrogens (xenoestrogens) are chemicals that bind to estrogen receptor, mimic estrogenic actions, and may have adverse effects on both human and wildlife health. Bisphenol A (BPA), a monomer used in the manufacture of epoxy resins and polycarbonate has estrogenic activity. In male rodents prenatal exposure to BPA resulted in modifications at the genital tract level. Our objective was to examine the effects of in utero exposure to low, environmentally relevant levels, of the xenoestrogen BPA on proliferation and differentiation of epithelial and stromal cells on the prepubertal rat ventral prostate. To characterize the periductal stromal cells phenotype the expression of vimentin and smooth muscle alpha-actin was evaluated. Androgen receptor (AR) and prostatic acid phosphatase (PAP) expression were also evaluated in epithelial and stromal compartments. Prenatal exposure to BPA increases the fibroblastic:smooth muscle cells ratio and decreases the number of AR-positive cells of periductal stroma of the ventral prostate. In contrast, no differences in AR expression were observed in epithelial cells between control and BPA-treated groups. No changes in proliferation patterns were observed in epithelial and stromal compartments; however, the expression of PAP was diminished in prostate ductal secretory cells of rats in utero exposed to BPA. Our results suggest that prenatal exposure to BPA altered the differentiation pattern of periductal stromal cells of the ventral prostate. These findings are significant in light of the data on human prostate cancers where alterations in the stroma compartment may enhance the invasive and/or malignant potential of the nascent tumor.     

Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation

At clinical concentrations, the potent intravenous general anesthetic etomidate enhances gamma-aminobutyric acid, type A (GABA(A)) receptor activity elicited with low gamma-aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA(A) receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channel-activating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(+)-etomidate isomer and display parallel dependence on GABA(A) beta subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human alpha1beta2gamma2L receptors. Etomidate at up to 100 microm reduced GABA EC(50) values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC(50) and increased maximal efficacy, demonstrating that etomidate shifts the GABA(A) receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux co-agonist mechanism with two equivalent etomidate sites that allosterically enhance GABA(A) receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA(A) receptors containing a point mutation that increases constitutive gating activity.     

A metabonomic study of inhibition of GABA uptake in the cerebral cortex

GABAergic activity is regulated by rapid, high affinity uptake of GABA from the synapse. Perturbation of GABA reuptake has been implicated in neurological disease and inhibitors of GABA transporters (GAT) have been used therapeutically but little detail is known about the ramifications of GAT inhibition on brain neurochemistry. Here, we incubated Guinea pig cortical tissue slices with [3-¹³C]pyruvate and major, currently available GABA uptake inhibitors. Metabolic fingerprints were generated from these experiments using ¹³C/¹H NMR spectroscopy. These fingerprints were analyzed using multivariate statistical approaches and compared with an existing library of fingerprints of activity at GABA receptors. This approach identified five distinct clusters of metabolic activity induced by blocking GABA uptake. Inhibition of GABA uptake via GAT1 produced patterns similar to activity at mainstream GABAergic synapses in particular those containing α1-subunits but still statistically separable. This indicated that inhibition of GABA uptake, an indirect method of activating GABA receptors, produces different effects to direct receptor activation or to exogenous GABA. The mechanism of inhibitor function also produced different outcomes, with the channel blocker SKF 89976A yielding a unique metabolic response. Blocking GAT1 and GAT3 simultaneously induces a large metabolic response consistent with induction of tonic inhibition via high affinity GABA receptors. Blocking BGT produces patterns similar to activity at less common receptors such as those containing α5 subunits. This approach is useful for determining where in the spectrum of GABAergic responses a particular GABA transport inhibitor is effective.     

Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors

The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.     

Cl(-) concentration changes and desensitization of GABA(A) and glycine receptors

Desensitization of ligand-gated ion channels plays a critical role for the information transfer between neurons. The current view on γ-aminobutyric acid (GABA)(A) and glycine receptors includes significant rapid components of desensitization as well as cross-desensitization between the two receptor types. Here, we analyze the mechanism of apparent cross-desensitization between native GABA(A) and glycine receptors in rat central neurons and quantify to what extent the current decay in the presence of ligand is a result of desensitization versus changes in intracellular Cl(-) concentration ([Cl(-)](i)). We show that apparent cross-desensitization of currents evoked by GABA and by glycine is caused by changes in [Cl(-)](i). We also show that changes in [Cl(-)](i) are critical for the decay of current in the presence of either GABA or glycine, whereas changes in conductance often play a minor role only. Thus, the currents decayed significantly quicker than the conductances, which decayed with time constants of several seconds and in some cells did not decay below the value at peak current during 20-s agonist application. By taking the cytosolic volume into account and numerically computing the membrane currents and expected changes in [Cl(-)](i), we provide a theoretical framework for the observed effects. Modeling diffusional exchange of Cl(-) between cytosol and patch pipettes, we also show that considerable changes in [Cl(-)](i) may be expected and cause rapidly decaying current components in conventional whole cell or outside-out patch recordings. The findings imply that a reevaluation of the desensitization properties of GABA(A) and glycine receptors is needed.     

Activation of central adenosine A(2A) receptors enhances superior laryngeal nerve stimulation-induced apnea in piglets via a GABAergic pathway.

Activation of the laryngeal mucosa results in apnea that is mediated through, and can be elicited via electrical stimulation of, the superior laryngeal nerve (SLN). This potent inhibitory reflex has been suggested to play a role in the pathogenesis of apnea of prematurity and sudden infant death syndrome, and it is attenuated by theophylline and blockade of GABA(A) receptors. However, the interaction between GABA and adenosine in the production of SLN stimulation-induced apnea has not been previously examined. We hypothesized that activation of adenosine A(2A) receptors will enhance apnea induced by SLN stimulation while subsequent blockade of GABA(A) receptors will reverse the effect of A(2A) receptor activation. The phrenic nerve responses to increasing levels of SLN stimulation were measured before and after sequential intracisternal administration of the adenosine A(2A) receptor agonist CGS (n = 10) and GABA(A) receptor blocker bicuculline (n = 7) in ventilated, vagotomized, decerebrate, and paralyzed newborn piglets. Increasing levels of SLN stimulation caused progressive inhibition of phrenic activity and lead to apnea during higher levels of stimulation. CGS caused inhibition of baseline phrenic activity, hypotension, and enhancement of apnea induced by SLN stimulation. Subsequent bicuculline administration reversed the effects of CGS and prevented the production of apnea compared with control at higher SLN stimulation levels. We conclude that activation of adenosine A(2A) receptors enhances SLN stimulation-induced apnea probably via a GABAergic pathway. We speculate that SLN stimulation causes endogenous release of adenosine that activates A(2A) receptors on GABAergic neurons, resulting in the release of GABA at inspiratory neurons and subsequent respiratory inhibition.