Gamma-aminobutyric Acid Type A Research Articles

Selective reduction of γ-aminobutyric acid type A receptor δ subunit mRNA levels by MK-801 in rat dentate gyrus

The influence of excitatory blockade elicited by uncompetitive N-methyl- d-aspartate (NMDA)/glutamate receptor antagonists on inhibitory GABAergic systems is not well understood. Adult male rats were injected i.p. with a single dose of the prototypical uncompetitive antagonist MK-801 (0.2–10 mg/kg) and in situ hybridization was performed to measure mRNA levels of γ-aminobutyric acid type A (GABA A) receptor subunits (α1–6, β1–3, γ1–3, δ, ε, and θ). A significant decrease in δ subunit mRNA levels, that reached ∼70% of saline-treated values, was observed in the hippocampal dentate gyrus following MK-801 administration. Other subunits did not display statistically significant alterations. These data demonstrate selective actions on GABA A receptor subunit levels that result from blockade of excitation by MK-801.

A TM2 Residue in the β1 Subunit Determines Spontaneous Opening of Homomeric and Heteromeric γ-Aminobutyric Acid-gated Ion Channels

Gamma-aminobutyric acid type A (GABAA) receptors are major inhibitory neurotransmitter-gated ion channels in the central nervous system. GABAA receptors consist of multiple subunits and exhibit distinct pharmacological and channel properties. Of all GABAA receptor subunits, the beta subunit is thought to be a key component for the functionality of the receptors. Certain types of GABAA receptors have been found to be constitutively active. However, the molecular basis for spontaneous opening of channels of these receptors is not totally understood. In this study, we showed that channels that contain the beta1 but not beta3 subunits opened spontaneously when these subunits were expressed homomerically or co-expressed with other types of GABAA receptor subunits in Xenopus oocytes. Using subunit chimeras and site-directed mutagenesis, we localized a key amino acid residue, a serine at position 265, that is critical in conferring an open state of the beta1 subunit-containing GABAA receptors in the absence of agonist. Moreover, some point mutations of Ser-265 also produced constitutively active channels. The magnitude of spontaneous activity of these receptors was correlated with the molecular volume of the residue at 265 for both homomeric and heteromeric GABAA receptors, suggesting that the spontaneous activity of the beta1 subunit-containing GABAA receptors may be mediated through a similar molecular mechanism that is dependent on the molecular volume of the residue at 265.

Specific GABA A Circuits for Visual Cortical Plasticity

Weak inhibition within visual cortex early in life prevents experience-dependent plasticity. Loss of responsiveness to an eye deprived of vision can be initiated prematurely by enhancing gamma-aminobutyric acid (GABA)-mediated transmission with benzodiazepines. Here, we use a mouse "knockin" mutation to alpha subunits that renders individual GABA type A (GABA(A)) receptors insensitive to diazepam to show that a particular inhibitory network controls expression of the critical period. Only alpha1-containing circuits were found to drive cortical plasticity, whereas alpha2-enriched connections separately regulated neuronal firing. This dissociation carries implications for models of brain development and the safe design of benzodiazepines for use in infants.

GABA A receptor abnormalities in Prader–Willi syndrome assessed with positron emission tomography and [ 11C]flumazenil

Prader–Willi syndrome (PWS) is a multi-system disorder characterized clinically by abnormal mental and physical development. PWS patients have a deletion in an imprinted region on paternal chromosome 15 (15q11–13), maternal disomy for this segment, or rarely, a chromosomal imprinting center deletion that gives rise to suppression of the equivalent paternal genes. Within the affected segment of chromosome 15 are genes encoding the α 5, β 3 and γ 3 subunits of the gamma-aminobutyric acid type-A (GABA A) receptor. Therefore, altered neurobehavioral function could arise in PWS due directly to altered GABA A receptor composition and expression, or alternatively, from brain developmental and maturational effects of these or other genes in the imprinted region. The aim of the present study was to assess cerebral GABA A receptors in PWS with the use of positron emission tomography of the benzodiazepine binding site employing [ 11C]flumazenil ([ 11C]FMZ). A reduction in [ 11C]FMZ binding was found predominantly in the cingulate, frontal and temporal neocortices and insula in six adult PWS patients compared to nine normal subjects. A possible role for the deleted β 3 subunit gene in PWS is supported in part by the wide cortical distribution of its mRNA expression and the effects of experimental knockouts on benzodiazepine binding described in prior studies. Altered GABA A receptor composition or number in these cortical regions may account for neurobehavioral abnormalities in PWS including mild mental retardation, poor impulse control, and impaired responses to somatic pain.

Cloning and Characterization of a GABAA Receptor γ2 Subunit Variant

We have cloned a novel gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit variant named gamma2XL. gamma2XL contains an alternatively spliced exon, resulting in the addition of 40 amino acids to the N-terminal extracellular domain between Ser171 and Tyr172. We show that gamma2XL failed to localize to the cell surface when it was coexpressed with the alpha2 and beta1 subunits in human embryonic kidney 293 cells. Expression of gamma2XL in 293 cells suppressed GABAA receptor binding in a dose-dependent manner by preventing GABAA receptor cell-surface localization. We also generated a gamma2 mutant with Ser171 and Tyr172 converted to glycine and threonine, respectively. We demonstrate that this mutant has a significantly lower affinity for the alpha2 and beta1 subunits and failed to reach the cell surface when coexpressed with these subunits. Together, our results indicate that Ser171 and Tyr172 in the gamma2 subunit constitute a critical motif. When this motif is disrupted by insertion of the alternative exon, access of the gamma2 subunit to the cell surface is prevented.

Dissociation of spikes, synaptic activity, and activity-dependent increments in rat cerebellar blood flow by tonic synaptic inhibition.

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF) to map the brain, but the physiological basis of the neuroimaging signals is still not well understood. Here we applied a pharmacological approach to separate spiking activity, synaptic activity, and the accompanying changes in CBF in rat cerebellar cortex. We report that tonic synaptic inhibition achieved by topical application of gamma-aminobutyric acid type A (GABAA) (muscimol) or GABAB (baclofen) receptor agonists abolished or reduced spontaneous Purkinje cell spiking activity without affecting basal CBF. The magnitude of CBF responses evoked by climbing fiber stimulation decreased gradually over time after exposure to muscimol, being more pronounced in the superficial than in the deep cortical layers. We provide direct evidence in favor of a laminar-specific regulation of CBF in deep cortical layers, independent of dilatation of surface vessels. With prolonged exposure to muscimol, activity-dependent CBF increments disappeared, despite preserved cerebrovascular reactivity to adenosine and preserved local field potentials (LFP). This dissociation of CBF and LFPs suggests that CBF responses are independent of extracellular synaptic currents that generate LFPs. Our work implies that neuronal and vascular signals evoked by glutamatergic pathways are sensitive to synaptic inhibition, and that local mechanisms independent of transmembrane synaptic currents adjust flow to synaptic activity in distinct cortical layers. Our results provide fundamental insights into the functional regulation of blood flow, showing important interference of GABAA receptors in translating excitatory input into blood flow responses.

Alcohol intoxication increases allopregnanolone levels in male adolescent humans.

Teenage drinking is a cause of growing concern in industrialized countries, where almost 35% of alcohol drinkers are under 16 years old. Increased anxiety, irritability and depression among adolescents may induce them to seek the anxiolytic and rewarding properties of alcohol. Allopregnanolone is rewarding in rodents, and therefore may contribute to the effects of alcohol. In this paper, we studied the effects of acute alcohol intoxication on the plasma levels of allopregnanolone in male adolescents. Blood samples were drawn from male adolescents who arrived at the Emergency Department of the Hospital. Two groups were studied: one study group was formed by adolescents who arrived with evident behavioral symptoms of acute alcohol intoxication (AAI) and the other by those arriving for mild trauma (contusions, sprains) after no consumption of alcohol (Controls). Our results demonstrate that AAI significantly increases serum allopregnanolone levels in male adolescents. Because alcohol and allopregnanolone positively modulate gamma-aminobutyric acid type A (GABAA) receptors, allopregnanolone may play a major role in the anxiolytic and rewarding effects of alcohol, either directly or by influencing the sensitivity of GABAA receptors to alcohol.

GABAA/alpha1 receptor agonists and antagonists: effects on species-typical and heightened aggressive behavior after alcohol self-administration in mice.

The positive modulation of gamma-aminobutyric acid type-A (GABAA) receptors is a putative mechanism via which alcohol escalates aggressive behavior. Broad-spectrum benzodiazepine antagonists block alcohol-heightened aggression in rats and monkeys. However, the degree to which GABAA subunit composition plays a role in heightened aggressive behavior induced by self-administration of a moderate alcohol dose remains unresolved. Beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) and zolpidem act preferentially at GABAA receptors containing the alpha1 subunit as antagonist and agonist, respectively, and serve as useful tools to evaluate the role of GABAA receptor subtypes in self-administered alcohol on aggression. Male resident mice, housed in breeding pairs, were conditioned to nose-poke in a removable panel in their home cage, with each fifth poke being reinforced by the delivery of 0.05 ml of 6% ethanol (EtOH). After consuming EtOH, the resident mice were given the antagonists beta-CCt and flumazenil or agonists zolpidem and triazolam, and then confronted an intruder male in their home cage for a 5-min period. Following self-administration of EtOH (1.0 g/kg, 1.7 g/kg), 14 of 37 resident mice displayed unusually large increases in the frequency of attack bites and sideways threats. Flumazenil or beta-CCt decreased alcohol-heightened and non-heightened aggression in a dose-dependent manner. Administration of 3 mg/kg beta-CCt lowered the aggression-heightening effects of 1 g/kg and 1.7 g/kg EtOH, but did not antagonize the sedative effects of 3.0 g/kg EtOH. Triazolam and zolpidem decreased alcohol-heightened and non-heightened aggressive behavior, and these antiaggressive effects were accompanied by reduced motor activity, indicating sedation. Benzodiazepine antagonists, particularly those acting preferentially at GABAA/alpha1 subunit-containing receptors, decrease alcohol-heightened and species-typical aggressive behavior, but are ineffective in attenuating the sedative effects of alcohol.

A Rapid Increase in the Total Number of Cell Surface Functional GABAA Receptors Induced by Brain-derived Neurotrophic Factor in Rat Visual Cortex

The number of postsynaptic gamma-aminobutyric acid type A (GABAA) receptors is a fundamental determinant of the variability of inhibitory synaptic responses in the central nervous system. In rat visual cortex, [3H]SR-95531 binding assays revealed that brain-derived neurotrophic factor (BDNF), one of the neurotrophins, induced a rapid increase in the total number of cell surface GABAA receptors, through the activation of Trk B receptor tyrosine kinases. We also demonstrated that BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons freshly isolated from P14 rats. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and phospholipase C-gamma. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. The selective increase in mean miniature inhibitory postsynaptic (mIPSC) current amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons. These results suggest that BDNF could alter the number of cell surface GABAA receptors in a region-specific manner.

A Single M1 Residue in the β2 Subunit Alters Channel Gating of GABAA Receptor in Anesthetic Modulation and Direct Activation

General anesthetics allosterically modulate the activity of neuronal gamma-aminobutyric acid, type A (GABAA), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of alpha and beta subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of beta2Gly-219 to Phe corresponded in rho nearly eliminated the modulatory effect of IV anesthetics in alpha1/beta2/gamma2S combination. However, the general anesthetics retained the ability to directly open the channel of mutant G219F, and the apparent affinity for GABA was increased, and desensitization rate was reduced. In this study, we made additional single mutations such as 219 Ser, Cys, Ile, Asp, Arg, Tyr, and Trp. The larger side chains of the replacement residues produced the greatest reduction in enhancement of GABA currents by IV anesthetics at clinical concentrations (Trp > Tyr = Phe > Arg > Asp > Ile > Cys > Ser = wild type). Compared with a 2-3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4- to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mm) and etomidate (100 mum) and for increased apparent affinity for direct gating by the IV anesthetics.

GABA and its agonists improved visual cortical function in senescent monkeys.

Human cerebral cortical function degrades during old age. Much of this change may result from a degradation of intracortical inhibition during senescence. We used multibarreled microelectrodes to study the effects of electrophoretic application of gamma-aminobutyric acid (GABA), the GABA type a (GABAa) receptor agonist muscimol, and the GABAa receptor antagonist bicuculline, respectively, on the properties of individual V1 cells in old monkeys. Bicuculline exerted a much weaker effect on neuronal responses in old than in young animals, confirming a degradation of GABA-mediated inhibition. On the other hand, the administration of GABA and muscimol resulted in improved visual function. Many treated cells in area V1 of old animals displayed responses typical of young cells. The present results have important implications for the treatment of the sensory, motor, and cognitive declines that accompany old age.

Three Cases of Zolpidem Dependence Treated with Fluoxetine: The Serotonin Hypothesis

Zolpidem is an Imidazopyridine hypnotic that is believed to act selectively at al subunit-containing gamma-aminobutyric acid type A (GABAA) receptors and thus to have minimal abuse and dependence potential. We present three cases of Zolpidem abuse and dependence in which the drug was used not for sedation but for stimulation and anxiolysis. All of the patients were treated with fluoxetine (a selective serotonin reuptake inhibitor) and managed to discontinue the abuse and remain abstinent from the drug. The efficacy of this kind of medication on the abuse of a GABAergic agonist, in this case dependence on zolpidem, leads to a serotonergic and GABAergic system interaction hypothesis.

Masking synchronous GABA-mediated potentials controls limbic seizures.

We determined how CA3-driven interictal discharges block ictal activity generated in the entorhinal cortex during bath application of 4-aminopyridine (4AP, 50 microM). Field potential and [K+]o recordings were obtained from mouse combined hippocampus-entorhinal cortex slices maintained in vitro. 4AP induced N-methyl-d-aspartate (NMDA) receptor-dependent ictal discharges that originated in the entorhinal cortex, disappeared over time, but were reestablished by cutting the Schaffer collateral (n = 20) or by depressing CA3 network excitability with local application of glutamatergic receptor antagonists (n = 5). In addition, two types of interictal activity occurred throughout the experiment. The first type was CA3 driven and was abolished by a non-NMDA glutamatergic receptor antagonist. The second type was largely contributed by gamma-aminobutyric acid type A (GABAA) receptor-mediated conductances and persisted during blockade of glutamatergic transmission. The absence of CA3-driven interictal discharges in the entorhinal cortex after Schaffer collateral cut facilitated the GABA-mediated interictal potentials that corresponded to large [K+]o elevations and played a role in ictal discharge initiation. Accordingly, ictal discharges along with GABA-mediated interictal potentials disappeared during GABAA-receptor blockade (n = 7) or activation of mu-opioid receptors that inhibit GABA release (n = 4). Our findings suggest that CA3-driven interictal events restrain ictal discharge generation in the entorhinal cortex by modulating the size of interictal GABA-mediated potentials that lead to large [K+]o elevations capable of initiating ictal discharges in this structure.

Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation.

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the gamma2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the gamma2 subunit interacts with itself as well as with gamma1, gamma3 and beta1-3 subunits, but not with the alpha subunits. We further show that gamma2 subunits interact with photolabeled pentameric GABAA receptors composed of alpha1, beta2/3 and gamma2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the gamma2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389-411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding gamma2-derived peptide. Using similar approaches, we identified the interaction motif in the gamma2 subunit mediating interaction with the beta2 subunit as a 47-amino acid motif that includes the gamma2 self-interacting motif. The identified gamma2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit-subunit interaction.

Molecular actions of (S)-desmethylzopiclone (SEP-174559), an anxiolytic metabolite of zopiclone.

This study set out to profile the activity of (S)-desmethylzopiclone (SEP-174559) at subtypes of the gamma-aminobutyric acid type-A (GABA(A)) receptor and other neurotransmitter receptor ion channels. Recombinant receptors were expressed in human embryonic kidney 293 cells and examined functionally by patch-clamp recording with fast perfusion of agonist and drug solutions. Micromolar concentrations of SEP-174559 potentiated GABA(A) receptor currents evoked by subsaturating concentrations of GABA. The potentiation was related to a leftward shift in the GABA dose-response curves, suggesting the drug acts to increase GABA binding affinity. The potentiation strictly required the presence of the gamma2 subunit; no enhancement was seen for receptors containing instead the gamma1 subunit or lacking a gamma subunit altogether. SEP-174559 and its parent compound, racemic zopiclone, were not selective between alpha1-, alpha2-, or alpha3-bearing GABA(A) receptors. Within the nicotinic receptor superfamily, SEP-174559 did not affect serotonin type-3 receptor function but was found to inhibit nicotinic acetylcholine (nACh) receptors. The inhibition of nACh receptors was noncompetitive and was mimicked by zopiclone, alprazolam, and diazepam. In the glutamate receptor superfamily, SEP-174559 inhibited N-methyl-D-aspartate (NMDA) receptor currents but did not affect non-NMDA receptors. These data confirm that SEP-174559 has benzodiazepine-like actions at gamma2-bearing subtypes of the GABA(A) receptor and suggest additional actions of benzodiazepine-site ligands at nACh and NMDA receptors.

Droperidol inhibits GABA(A) and neuronal nicotinic receptor activation.

Droperidol is used in neuroleptanesthesia and as an antiemetic. Although its antiemetic effect is thought to be caused by dopaminergic inhibition, the mechanism of droperidol's anesthetic action is unknown. Because gamma-aminobutyric acid type A (GABAA) and neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as putative targets of other general anesthetic drugs, the authors tested the ability of droperidol to modulate these receptors. gamma-Aminobutyric acid type A alpha1beta1gamma2 receptor, alpha7 and alpha4beta2 nAChRs were expressed in Xenopus oocytes and studied with two-electrode voltage clamp recording. The authors tested the ability of droperidol at concentrations from 1 nm to 100 microm to modulate activation of these receptors by their native agonists. Droperidol inhibited the GABA response by a maximum of 24.7 +/- 3.0%. The IC50 for inhibition was 12.6 +/- 0.47 nm droperidol. At high concentrations, droperidol (100 microm) activates the GABAA receptor in the absence of GABA. Inhibition of the GABA response is significantly greater at hyperpolarized membrane potentials. The activation of the alpha7 nAChR is also inhibited by droperidol, with an IC50 of 5.8 +/- 0.53 microm. The Hill coefficient is 0.95 +/- 0.1. Inhibition is noncompetitive, and membrane voltage dependence is insignificant. Droperidol inhibits activation of both the GABAA alpha1beta1gamma2 and alpha7 nAChR. The submaximal GABA inhibition occurs within a concentration range such that it might be responsible for the anxiety, dysphoria, and restlessness that limit the clinical utility of high-dose droperidol anesthesia. Inhibition of the alpha7 nAChR might be responsible for the anesthetic action of droperidol.

Anesthetic properties of 4-iodopropofol: implications for mechanisms of anesthesia.

Positive modulation of gamma-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative-anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor-dependent and -independent anesthetic actions. The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA alpha1beta2gamma2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel-mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 +/- 0.5 microM) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg). Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.

Altered GABAA Receptor Subunit and Splice Variant Expression in Rats Treated With Chronic Intermittent Ethanol

Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Forskolin modulation of desensitization at GABA(A) and glycine receptors is not mediated by cAMP-dependent protein kinase in isolated carp amacrine-like cells.

The effects of forskolin on gamma-aminobutyric acid type-A (GABA(A)) and glycine receptors in amacrine-like cells of carp (Carassius auratus) retina were studied using patch-clamp techniques. Application of 50 microM forskolin markedly accelerated the desensitization of whole-cell responses induced by 100 microM GABA or glycine without changing the peak amplitude of the response. Both 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and 3-isobutyl-1 -methylxanthine (IBMX) (500 microM) failed to accelerate the desensitization of these two receptors. Protein kinase A (PKA) inhibitors, N- [2-[(p-bromocinnamyl)amino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89) and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8), could not block these effects of forskolin. An inactive analogue of forskolin, 1,9-dideoxyforskolin (DFSK), accelerated the desensitization effectively. These results suggest that forskolin's effects are not mediated by activation of the PKA pathway. Moreover, similar results were obtained using excised outside-out patches of these cells, suggesting that forskolin may act on an extracellular site(s). The neurosteroids 5alpha-pregnane-3alpha,21 -diol-20-one (THDOC) and 5-pregnen-31 -ol-20-one sulfate sodium (PS), structural analogues of forskolin, accelerated the desensitization of these receptors without changing the peak amplitudes, thus mimicking forskolin's effects. Furthermore, PS interacted with forskolin on these receptors so as to slow down the responses. These results raise the possibility that forskolin acts directly on an extracellular site(s) of the GABA(A) and glycine receptors, shared with neurosteroids, in carp amacrine-like cells.

Agent-selective effects of volatile anesthetics on GABAA receptor-mediated synaptic inhibition in hippocampal interneurons.

A relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal cells in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role for the maintenance of anesthesia. The aim of this study was to compare effects produced by volatile anesthetics on inhibitory postsynaptic currents (IPSCs) of rat hippocampal interneurons. Pharmacologically isolated gamma-aminobutyric acid type A (GABAA) receptor-mediated IPSCs were recorded with whole cell patch-clamp techniques in visually identified interneurons of rat hippocampal slices. Neurons located in the stratum radiatum-lacunosum moleculare of the CA1 region were studied. The effects of clinically relevant concentrations (1.0 rat minimum alveolar concentration) of halothane, enflurane, isoflurane, and sevoflurane were compared on kinetics of both stimulus-evoked and spontaneous GABAA receptor-mediated IPSCs in interneurons. Halothane (1.2 vol% approximately 0.35 mm), enflurane (2.2 vol% approximately 0.60 mm), isoflurane (1.4 vol% approximately 0.50 mm), and sevoflurane (2.7 vol% approximately 0.40 mm) preferentially depressed evoked IPSC amplitudes to 79.8 +/- 9.3% of control (n = 5), 38.2 +/- 8.6% (n = 6), 52.4 +/- 8.4% (n = 5), and 46.1 +/- 16.0% (n = 8), respectively. In addition, all anesthetics differentially prolonged the decay time constant of evoked IPSCs to 290.1 +/- 33.2% of control, 423.6 +/- 47.1, 277.0 +/- 32.2, and 529 +/- 48.5%, respectively. The frequencies of spontaneous IPSCs were increased by all anesthetics (twofold to threefold). Thus, the total negative charge transfer mediated by GABAA receptors between synaptically connected interneurons was enhanced by all anesthetics. Volatile anesthetics differentially enhanced GABAA receptor-mediated synaptic inhibition in rat hippocampal interneurons, suggesting that hippocampal interneuron circuits are depressed by these anesthetics in an agent-specific manner.