High affinity, heterogeneous displacement of [3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists

Heterogeneous binding interactions of cerebellar GABA(A) receptors were investigated with GABA agonists and neurosteroids. GABA(A) receptors of rat cerebellum were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [(3)H]EBOB binding. Both GABA and 5alpha-tetrahydrodeoxycorticosterone (5alpha-THDOC) displaced [(3)H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 microM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5alpha-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 microM ) and 5alpha-THDOC (0.3 microM ) potentiated the micromolar population of displacement by the other. Taurine inhibited [(3)H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 microM 5alpha-THDOC potentiated this inhibition. 5beta-THDOC did not affect [(3)H]EBOB binding significantly but in 1 microM it antagonised selectively the nanomolar displacement by 5alpha-THDOC. [(3)H]EBOB binding to hippocampal GABA(A) receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open alpha(6)betadelta GABA(A) receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.     

A 17beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA A receptors with nanomolar affinity

BACKGROUND AND PURPOSE: High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. EXPERIMENTAL APPROACH: Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. KEY RESULTS: Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. CONCLUSIONS AND IMPLICATIONS: HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.     

GABAA receptor open-state conformation determines non-competitive antagonist binding

The γ-aminobutyric acid (GABA) type A receptor (GABA_AR) is one of the most important targets for insecticide action. The human recombinant β3 homomer is the best available model for this binding site and 4-n-[³H]propyl-4′-ethynylbicycloorthobenzoate ([³H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the β3 homomer relative to the much-less-active but structurally very-similar β1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The β1 and β3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera β3/β1 with the β3 subunit extracellular domain and the β1 subunit transmembrane helices retained the high [³H]EBOB binding level of the β3 homomer while chimera β1/β3 with the β1 subunit extracellular domain and the β3 subunit transmembrane helices had low binding activity similar to the β1 homomer. GABA at 3 μM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive β1/β3 chimera close to wildtype α1β1 activity. EBOB binding was significantly altered by mutations β1S15′N and β3N15′S compared with wildtype β1 and β3, respectively. However, the binding activity of α1β1S15′N was insensitive to GABA and α1β3N15′S was stimulated much less than wildtype α1β3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation β3N15′S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA_A receptor sensitivity.     

In vivo labelling of alpha5 subunit-containing GABA(A) receptors using the selective radioligand [(3)H]L-655,708

L-655,708 is an imidazobenzodiazepine possessing 30-70-fold selectivity for the benzodiazepine binding site of GABA_A receptors containing an α5 rather than α1, α2 or α3 subunit. In the present study, [³H]L-655,708 was used to label mouse brain benzodiazepine binding sites in vivo. When compared to inhibition of in vivo binding of the non-selective ligand [³H]Ro 15-1788, the pharmacology of mouse in vivo [³H]L-655,708 binding was consistent with selective in vivo labelling of α5 subunit-containing GABA_A receptors. Thus, diazepam was equipotent at inhibiting in vivo [³H]L-655,708 and [³H]Ro 15-1788 binding; zolpidem, which has very low affinity for α5-containing GABA_A receptors, gave no inhibition of in vivo [³H]L-655,708 binding despite inhibiting in vivo [³H]Ro 15-1788 binding; and L-655,708 was more potent at inhibiting the in vivo binding of [³H]L-655,708 compared to [³H]Ro 15-1788. This pharmacological specificity of in vivo [³H]L-655,708 binding was confirmed autoradiographically. Hence, the anatomical distribution of in vivo [³H]L-655,708 binding was comparable to the distribution of α5-containing GABA_A receptors identified in vitro. Moreover, this distribution was distinct from that identified using [³H]Ro 15-1788. These data therefore suggest that [³H]L-655,708 can be used to identify α5-containing GABA_A receptors in vivo and that this ligand can be used to measure receptor occupancy of α5-selective ligands.     

Differential effects of short- and long-term zolpidem treatment on recombinant α1β2γ2s subtype of GABAA receptors in vitro

Aim:

Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABA_A receptors, which is increasingly prescribed. Recent studies suggest that prolonged zolpidem treatment induces tolerance. The aim of this study was to explore the adaptive changes in GABA_A receptors following short and long-term exposure to zolpidem in vitro.

Methods:

Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1β2γ2s GABA_A receptors were exposed to zolpidem (1 and 10 μmol/L) for short-term (2 h daily for 1, 2, or 3 consecutive days) or long-term (continuously for 48 h). Radioligand binding studies were used to determine the parameters of [³H]flunitrazepam binding sites.

Results:

A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [³H]flunitrazepam binding sites nor the affinity. In both control and short-term zolpidem treated groups, addition of GABA (1 nmol/L–1 mmol/L) enhanced [³H]flunitrazepam binding in a concentration-dependent manner. The maximum enhancement of [³H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group. In contrast, long-term exposure to zolpidem resulted in significantly increase in the maximum number of [³H]flunitrazepam binding sites without changing the affinity. Furthermore, long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [³H]flunitrazepam binding.

Conclusion:

The results suggest that continuous, but not intermittent and short-term, zolpidem-exposure is able to induce adaptive changes in GABA_A receptors that could be related to the development of tolerance and dependence.     

Neurosteroids may differentially affect the function of two native GABAA receptor subtypes in the rat brain

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA_A receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA_A receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3α,5α-P), at nanomolar concentrations, potentiated the GABA-induced [³H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10μM. The enhancement by GABA of the K⁺-evoked [³H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3α,5α-P; in addition, at 1–10μM, the steroid increased [³H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3α,5α-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10μM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [³H]-D-aspartate release elicited by 3μM 3α,5α-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3α,5α-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA_A receptors under study. Low micromolar 3α,5α-P can directly activate the cerebellar receptor, whereas the hippocampal GABA_A receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA_A receptor having distinct pharmacology, neuronal localization and function. Received: 13 October 1997 / Accepted: 30 December 1997     

Neonatal administration of a GABA-T inhibitor alters central GABAA receptor mechanisms and alcohol drinking in adult rats

Long-term effects of chronic treatment with a GABA-T (GABA-transaminase) inhibitor, ethanolamine O-sulphate (EOS) (200 mg/kg/day for the postnatal days 3–21) on the binding parameters of GABA_A receptors, hypothalamic monoamines and subsequent behavior were studied in Wistar rats. At the age of 1 month, EOS-treated rats showed reduced activity in the open-field and, at the age of 4 months, their voluntary alcohol consumption was increased. No changes were seen in Porsolt's swim test or in the plus-maze test. Weight gain was significantly retarded in EOS-treated rats. Maximal stimulation of [³H] flunitrazepam binding by GABA was decreased in the cerebral cortex and the EC₅₀-value for the GABA stimulation increased in the hippocampus in the EOS rats at the age of 4 months. EOS treatment did not alter the cerebellar diazepam sensitive and insensitive binding components of the imidazobenzodiazepine [³H]Ro 15-4513. No changes were observed in the hypothalamic monoamine concentrations. The results are in agreement with the idea that GABA-T inhibitor treatment permanently alters GABA_A mechanisms. Moreover, altering the CNS GABA level during development increases adult alcohol intake in rat.     

Pyrazolopyridines: Effect of structural alterations on activity at adenosine- and GABAA-receptors

A series of 4-substituted 1H-pyrazolo[3,4-b]pyridine- 5-carboxylic acid derivatives related in structure to the putative anxiolytics cartazolate, tracazolate, and etazolate were assessed for affinity at A₁- and A_2A-adenosine receptors and at GABA-, benzodiazepine-, and picrotoxinin- binding sites of the GABA_A-receptor-channel. None of the derivatives had markedly greater affinity at A₁-receptors than cartazolate (K_i-0.5 μM), but many had markedly lower affinity than cartazolate (K_i-1.5 μM) at A_2A-receptors. At the benzodiazepine-binding site of GABA_A-receptors some of the derivatives enhanced [³H]diazepam binding, as did cartazolate and GABA, some had no effect and some inhibited binding. Most of the derivatives inhibited binding of the benzodiazepine-antagonist [³H]Ro 15-1788. At the GABA-binding site, only a few of the derivatives inhibited binding of the antagonist [³H]SR-95531, as did GABA. At the picrotoxinin-binding site, many inhibited binding of [³⁵S]TBPS, but none were as potent as cartazolate or GABA. Analysis of the interactions indicates that stimulation of [³H]diazepam binding is allosteric and results from binding of the pyrazolopyridine at the GABA site or a subdomain of that site, while inhibition of [³H]Ro 15-1788 binding is competitive and due to binding at the benzodiazepine site. Inhibition of [³⁵S]TBPS binding at the picrotoxinin-channel site appears to be allosteric through the GABA site and/or by direct competition at the channel site. Alterations in structure markedly alter the affinities of pyrazolopyridines at such sites on the GABA_A-receptor-channel. Drug Dev. Res. 42:41–56, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Alterations in brain monoamines and GABAA receptors in transgenic mice overexpressing TGFα

This study investigated the possibility that overexpression of transforming growth factor α (TGFα) changes those neurotransmitter systems that have been associated with behaviors found to be altered in the transgenic TGFα CD-1 mice. The female TGFα mice showed elevated levels of norepinephrine (NE) in the hypothalamus and serotonin (5-HT) in the cortex and brain stem when compared with nontransgenic CD-1 females. The concentrations of monoamines were not altered in the male transgenic brain. The 5-hydroxyindoleacitic acid (5-HIAA)/5-HT ratio was significantly reduced in the brain stem of the male TGFα mice and frontal cortex in the female transgenics. The binding of the [³H]GBR 12935-labeled DA transporter was lower in the frontal cortex in the transgenic male TGFα mice than in the female TGFα mice. No gender difference in dopamine (DA) transporter binding was noted between the nontransgenic male and female mice. Serotonin and GABA_A receptors were measured only in males. No differences in the number of 5-HT_1A and 5-HT₂ receptors were found in the cortex or hippocampus. Maximal GABA stimulation of [³H]flunitrazepam binding in the forebrain hemispheres and cerebellar binding of an imidazobenzodiazepine, [³H]Ro 15-4513, were not different between transgenic and nontransgenic male mice. However, forebrain [³⁵S]TBPS binding in male TGFa mice was less affected by the blockade of the GABA agonist sites by the specific GABA_A antagonists SR 95531 and bicuculline than the binding of the controls, suggesting either altered endogenous GABA concentrations or a change in receptor populations. Taken together, the previously reported behavioral alterations in male TGFa mice, including increased levels of aggressive behavior, locomotor activity, voluntary alcohol consumption, and immobility in the swim test, or the altered behavioral responses to alcohol and monoamine uptake inhibitors, may be due to a reduced 5-HIAA/5-HT ratio, [³H]GBR 12935-labeled DA transporter binding, or altered regulation of [³⁵S]TBPS binding by endogenous GABA in the brain. Reduced aggressive behavior and shortened immobility in the swim test in the female TGFa mice, on the other hand, might reflect elevated levels of NE and 5-HT in the brain. It is possible that TGFα-induced increase in plasma estrogen levels in the transgenic mice is the common mechanism of action that causes gender-specific changes in certain neurotransmitter systems.     

The plasma–occupancy relationship of the novel GABAA receptor benzodiazepine site ligand, α5IA,is similar in rats and primates

Background and purpose:

α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1-, α2-, α3- and α5-containing GABA_A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey).

Experimental approach:

In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg⁻¹) using an in vivo {[³H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[¹²³I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} γ-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC₅₀) was calculated.

Key results:

In rats, α5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC₅₀ was time-independent, ranging from 42 to 67 ng·mL⁻¹ over a 24 h time course with the average being 52 ng·mL⁻¹ (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC₅₀ for α5IA displacing steady-state [¹²³I]iomazenil binding was 57 ng·mL⁻¹.

Conclusions and implications:

Rat plasma EC₅₀ values did not vary as a function of time indicating that α5IA dissociates readily for the GABA_A receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [³H]flumazenil in vivo binding in rats and [¹²³I]iomazenil γ-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC₅₀ values (52 and 57 ng·mL⁻¹ respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.     

Failure ofγ-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex

The present study was designed to evaluate the possible interaction of-hydroxybutyrate (GHB) with the GABA_A receptor complex in the rat cerebral cortex. To this purpose we studied the effect of in vitro addition and in vivo administration of GHB on the biochemical parameters currently used to evaluate the function of the GABAergic system. In vitro addition of increasing concentrations of GHB failed to modify [³H]flunitrazepam (³H]FNZ) binding and the modulatory action of GABA on this binding. Moreover, unlike diazepam, GHB did not modify in vitro both muscimol-stimulated³⁶Cl^– uptake and t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat cerebral cortex. In vivo administration of sedative and hypnotic doses of GHB (300–750 mg/kg IP) failed to induce in 60 min any significant change in the [³⁵S]TBPS binding to unwashed cortical membranes. Moreover, GHB also failed to antagonize the increase in [³⁵S]TBPS binding (+55%) induced by isoniazid (350 mg/kg SC). In contrast, at the highest doses used, this drug completely antagonized the seizure activity induced by isoniazid. In conclusion, our data show that GHB fails to alter the function of the GABA_A/benzodiazepine/ionophore receptor complex in the rat cerebral cortex.     

3-Hydroxy-2'-methoxy-6-methylflavone: a potent anxiolytic with a unique selectivity profile at GABA(A) receptor subtypes

Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA_A receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA_A receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC₅₀ values between 38 and 106 μM) and occurred in a flumazenil-insensitive manner. 3-OH-2′MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4–11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2′MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors (EC₅₀ values between 1.4 and 2.5 μM) was 10-fold higher than at α4β1δ GABA_A receptors. 3-OH-2′MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2′MeO6MF (1–100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2′MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA_A receptor subtypes.     

Thermodynamics and kinetics of t-butylbicyclophosphorothionate binding differentiate convulsant and depressant barbiturate stereoisomers acting via GABAA ionophores

The temperature dependence of [³⁵S]-t- butylbicyclophosphorothionate (TBPS) binding to the convulsant sites of the GABA_A receptor complex was studied in membrane preparations of rat forebrain. Although specific [³⁵S]TBPS binding was maximal around 20° C, the rate constants of dissociation decreased monotonously between 37°C and 2° C. The displacing potencies of the convulsant S(+) enantiomer of 1-methyl-5-phenyl-5-propyl-barbituric acid (MPPB) (IC₅₀ = 1250 ± 30 M) and the depressant R(–) MPPB (IC_5O = 310 ± 5 M) did not show significant changes between 19° C and 37° C. Therefore barbiturate binding seems to be driven by entropic, rather than enthalpic changes. An excess of MPPB enantiomers elicited accelerated and polyphasic dissociations of [³⁵S]TBPS as compared to the monophasic dissociation by TBPS. Arrhenius analysis was applied to the measurable initial rate constants of dissociation. Arrhenius plots were linear between 2° C and 37° C. Activation parameters were similar when [³⁵S] TBPS dissociation was triggered by the convulsants TBPS and S(+) MPPB. It can be attributed to similar conformations of the closed ionophore complex. In contrast, the depressant R(–) MPPB strongly decreased the activation energy of TBPS dissociation from the open ionophore ternary complex.In whole-cell patch-clamp experiments R(–) MPPB, but not S(+) MPPB, elicited chloride currents in rat primary cortical cultures with an EC₅₀ value of 560 ± 30 M and a Hill coefficient of 2.9 ± 0.2. These currents were similar to those elicited by GABA and blocked by TBPS. A kinetic scheme is proposed for the dissociation of TBPS and to explain the different effects of MPPB enantiomers. Submillimolar R(–) MPPB is supposed to bind to (about three) barbiturate sites on GABA_A-ionophores and to open them in a cooperative manner to result in a decreased activation energy for accelerated displacement of convulsant binding.     

Is upregulation of benzodiazepine receptors a compensatory reaction to reduced GABAergic tone in the brain of stressed mice?

Effects of various forms of stress on the GABA_A receptor-chloride ionophore complex in the brain of NMRI mice were investigated. Male albino mice were subjected to stress by placing them on small platforms (SP; 3.5 cm diameter) surrounded by water for 24 h. This experimental model contains several stress factors like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. As additional stress control groups we used animals subjected to isolation, large platform (9.0 cm diameter) and repeated swimming stress. SP stress induced an increase in the number of cortical benzodiazepine (BDZ) receptors and a reduction in the GABA-stimulated ³⁶C1^– uptake by brain microsacs, whereas none of these changes could be observed in animals exposed to isolation, swimming or large platform stresses. Furthermore, the amount of GABA-induced stimulation of [³H]flunitrazepam binding was reduced in cortical brain membranes of SP-stressed animals, an effect due to fact that these animals dispayed an increase in the basal [³H]flunitrazepam binding, whereas the absolute level of maximally enhanced BDZ binding in the presence of GABA did not differ from those found in controls. Neither basal [³H]muscimol binding or thiopentone sodium-induced stimulation of [³H]flunitrazepam binding were changed in any group of stressed mice. It is proposed that the observed upregulation in the number (B _max ) of cortical BDZ receptors in SP-stressed mice may represent a compensatory response to a stress-induced attenuation of GABAergic neurotransmission.     

Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABA_A receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA_A receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA_A receptors. α4/δ-Containing GABA_A receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀ = 24 nM) and α4β3δ (EC₅₀ = 12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1) = 16 nM; EC₅₀(2) = 1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀ = 1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA_A receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA_A receptors compared to their synaptic counterparts.     

α5 Subunit-containing GABAA receptors mediate a slowly decaying inhibitory synaptic current in CA1 pyramidal neurons following Schaffer collateral activation

GABA_A receptors that contain the α5 subunit (α5GABA_ARs) are highly expressed in the hippocampus, and have been implicated in learning and memory processes. They generate a tonic form of inhibition that regulates neuronal excitability. Recently it was shown that α5GABA_ARs also contribute to slow phasic inhibition of CA1 pyramidal neurons following local stimulation in the stratum lacunosum moleculare. However, it is unknown whether α5GABA_ARs can also be recruited indirectly by stimulation of Schaffer collaterals. Here, we studied GABAergic currents evoked by stimulation in the stratum radiatum of CA1 in the presence and absence of CNQX to block AMPA receptor-mediated excitation. We tested their sensitivity to gabazine and two drugs acting at the benzodiazepine site of α1/α2/α3 or α5GABA_ARs (400 nM zolpidem and 20 nM L-655,708, respectively). IPSCs evoked by stimulation in the stratum radiatum in the presence of CNQX were potentiated by zolpidem, blocked by 1 μM gabazine and were relatively insensitive to L-655,708 consistent with the lack of α5GABA_ARs. In contrast, IPSCs evoked by stimulation of Schaffer collaterals had a significant gabazine-insensitive component. This component was attenuated by L-655,708 and enhanced by burst stimulation. Furthermore, the L-655,708-sensitive current was absent in recordings from mice lacking α5GABA_ARs (gabra5^−/− mice). These results show that α5GABA_AR-mediated phasic inhibition is activated by the Schaffer collateral pathway and provide evidence for activity pattern-dependent participation of α5GABA_ARs in inhibition.     

6-Methylflavone,a benzodiazepine receptor ligand with antagonistic properties on rat brain and human recombinant GABAA receptors in vitro

Seventeen flavonoids were found to have inhibitory activity on the central nervous system GABA_A/benzodiazepine (BZD) receptors with IC₅₀ values ranging from 0.12 to 8 μM. 6-Methylflavone, the most potent, was pharmacologically characterized by radioligand binding assays on rat brain membranes in vitro and human recombinant GABA_A/BZD receptors expressed in Sf-9 insect cells, as well as electrophysiologically by the whole-cell patch clamp technique. Scatchard plot analysis showed that 6-methylflavone was a competitive inhibitor of [³H]-Ro 15-1788, binding to rat brain cortical membranes. The GABA ratio of 1.06 for [³H]-diazepam binding to cortex and 1.23 for cerebellum indicated an antagonistic or a weak partial agonistic profile of 6-methylflavone on the rat BZD₁ receptors, while the GABA ratio of 0.76 on hippocampus indicated an antagonistic or partial-inverse agonistic profile on the BZD₂ receptors. In Sf-9 insects cells, the GABA ratios showed a weak partial agonistic profile on the α₁β₂γ_2S (GABA ratio 1.29) subtype combination, an antagonistic profile on the α₂β₂γ_2S (1.13) and α₃β₂γ_2S (1.03), and a partial inverse agonistic profile on the α₅β₂γ_2S (0.79) subtype combination. The modulation of GABA-induced chloride currents by 6-methylflavone suggests that the compound is an antagonist at human GABA_A receptor subtypes. Based on our data of GABA_A/BZD receptor active as well as inactive flavonoids, some general structure–activity relationships are discussed. Drug Dev. Res. 41:99–106, 1997. © 1997 Wiley-Liss, Inc.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

Allosteric modulation of 5-HT(1A) receptors by zinc: Binding studies

5-HT_1A receptors were studied via [³H]WAY-100635 and [³H]8-OH-DPAT binding to rat brain cortical membranes. We characterized the effect of zinc (Zn²⁺) on the binding properties of the 5-HT_1A receptor. The allosteric ternary complex model was applied to determine the dissociation constant (K_A) of Zn²⁺ and their cooperativity factors (α) affecting the dissociation constants (K_D, K_i) of [³H]WAY-100635, [³H]8-OH-DPAT, and serotonin (5-HT), the endogenous neurotransmitter. Zn²⁺ (5 μM-1 mM) inhibited the binding of agonist/antagonist to 5-HT1A receptors, mostly by decreasing both the ligands' affinity and the maximal number of sites. In [³⁵S]GTPγS binding assays Zn²⁺ behaved as insourmountable antagonist of 5-HT1A receptors, in agreement with radioligand binding assays. The residues involved in the formation of the inhibitory binding site on the 5-HT1A receptor were assessed by using N-ethyl-maleimide (NEM) or diethylpyrocarbonate (DEPC) which modify preferentially cysteine and histidine residues, respectively. Exposure to both agents did not block the negative allosteric effects of Zn²⁺ on agonist and antagonist binding. Our findings represent the first quantitative analysis of allosteric binding interactions for 5-HT_1A receptors. The physiological significance of Zn²⁺ modulation of 5-HT_1A receptors is unclear, but the colocalization of 5-HT_1A receptors and Zn²⁺ in the nervous system (e.g. in the hippocampus and cerebral cortex) suggests that Zn²⁺ released at nerve terminals may modulate signals generated by the 5-HT_1A receptors in vivo. Finally, these findings suggest that synaptic Zn²⁺ may be a factor influencing the effectiveness of therapies that rely on 5-HT_1A receptor activity.