Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain γ-aminobutyric acid_A (GABA_A) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABA_A-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABA_A-receptors would also exhibit a differential response to halothane as assayed by an in vitro³⁶Cl⁻ influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 μmol/1 GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC₅₀ 336±64 μmol/1 (S.E.M.) vs. 605±110 μmol/1, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7±0.4 nmol·mg⁻¹·3⁻¹, vs. DR 4.7±0.5nmol·mg⁻¹·3s⁻). Halothane (500 μmol/1) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC₅₀ for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC₅₀ 1.8±0.4 μmol/1vs.14.7±0.9 μmol/1 without halothane) than in the DR mice (GABA EC₅₀ 4.7±0.6 μmol/1vs.14.7±0.9 μmol/1 without halothane). This effect of halothane was highly significant, both when compared to control, and between the selected lines (P < 0.001). The findings that halothane enhances GABA-gated flux and enhances GABA's channel gating potency support the hypothesis that differential enhancement of agonist-stimulated chloride permeability at GABA_A receptors could be a mechanism underlying the differential obtunding potency of halothane in DS and DR mice. However, at high GABA concentrations halothane decreased maximal chloride flux, more in DS than in DR mice (P < 0.001), which is not consistent with such a mechanism.     

A quantitative relationship between Cl- enhanced [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate binding to the benzodiazepine/GABA receptor chloride ionophore complex

A quantitative relationship between the efficacy (i.e. maximum enhancement) of Cl- to increase [3H]flunitrazepam binding and the density of [35S]t-butylbicyclophosphorothionate binding sites was observed in well-washed membrane fragments of rat cerebral cortex previously exposed to phospholipase A2. This relationship (described by the equation y = ABx) was maintained when [3H]flunitrazepam was assayed at Cl- concentrations between 100 and 600 mM, and was not qualitatively altered by the presence of 100 microM pentobarbital. However, under experimental conditions that reduced the ratio of [35S]TBPS binding sites/benzodiazepine receptors, the effects of pentobarbital suggest that the conductance state of benzodiazepine receptor-coupled chloride channels may be the primary determinant of Cl- enhanced [3H]flunitrazepam binding.     

Pregnenolone-sulfate: an endogenous antagonist of the gamma-aminobutyric acid receptor complex in brain?

The interaction of the 'neurosteroid', pregnenolone-sulfate (PS), with the GABA/benzodiazepine/chloride ionophore receptor complex was investigated in rat brain subcellular preparations. At low micromolar concentrations PS competitively inhibited the binding of the convulsant [35S]t-butylbicyclophosphorothionate (TBPS) and antagonized pentobarbital-stimulated [3H]flunitrazepam binding to synaptosomes. In addition, PS inhibited muscimol-stimulated 36Cl-uptake in brain synaptoneurosomes, including that PS has characteristics of a relatively potent antagonist of the chloride channel coupled to the GABA receptor. Together with our previous finding that A-ring reduced metabolites of progesterone and deoxycorticosterone also interact with the GABA receptor complex but as hypnotic barbiturates, these data suggest that the regulation of GABAergic neurotransmission by various neurosteroids may be an important mechanism for controlling neuronal excitability.     

gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons.

Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [3H]flunitrazepam binding was rapid and saturable, with an apparent Kd of 4.2 +/- 1.5 nM and Bmax of 776 +/- 54 fmol/mg protein. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC50 = 9 +/- 2 microM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [3H]flunitrazepam, [3H]GABA, [3H]Ro15-1788, [3H]Ro15-4513 and [35S]TBPS binding, a reduced enhancement of [3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABAA-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABAA receptor-mediated slow process.     

Characterization of antagonistic activity and binding properties of SR 95531, a pyridazinyl-GABA derivative, in rat brain and cultured cerebellar neuronal cells.

Experiments were performed to characterize the antagonistic activity and binding properties of SR 95531 [2-(3' carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-piridazinium bromide] in rat brain. SR 95531 and bicuculline methiodide inhibited muscimol-stimulated 36Cl- uptake in cortical synaptoneurosomes in a concentration-dependent manner. The inhibitory potency of SR 95531 for the muscimol-stimulated 36Cl- uptake was 15 times higher than that of bicuculline methiodide. Scatchard plots of binding isotherms exhibited two apparent binding sites for [3H]SR 95531 in both the frontal cortex and cerebellum. The IC50 value of SR 95531 for muscimol-stimulated 36Cl- uptake into cortical synaptoneurosomes was in close agreement with the KD value of low-affinity binding sites of [3H]SR 95531 in the frontal cortex. Pretreatment of the membranes with phospholipase A2 invariably decreased [3H]SR 95531 binding in the frontal cortex and cerebellum. On the other hand, the treatment significantly increased [3H]gamma-aminobutyric acid (GABA) binding in a concentration-dependent manner in the frontal cortex. Although lower concentrations of phospholipase A2 did not affect [3H]GABA binding in the cerebellum, treatment with higher concentrations of phospholipase A2 increased the binding in this region. Specific binding of [3H]SR 95531 was also detected in cultures rich in cerebellar granule cells. Pretreatment with phospholipase A2 affected the binding of [3H]GABA and [3H]SR 95531 in these cells, as in the case of the cerebellum. These effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531 were partially prevented by the addition of delipidated bovine serum albumin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Differential seizure sensitivities to picrotoxinin in two inbred strains of mice (DBA/2J and BALB/c ByJ): parallel changes in GABA receptor-mediated chloride flux and receptor binding

Two strains of mice were shown to possess a differential sensitivity to picrotoxinin-induced convulsions; picrotoxinin elicited both tonic and clonic seizures at lower doses in the DBA/2J (DBA) strain compared to the BALB/c ByJ (BALB) strain. Less protection of picrotoxinin-induced tonic seizures was afforded by pentobarbital in the DBA strain. Biochemical studies revealed that picrotoxin inhibited 36Cl- efflux from forebrain synaptoneurosomes only in the DBA strain. In addition, picrotoxin inhibited pentobarbital-induced 36Cl- efflux to a greater extent in the DBA strain. No differences were observed in the binding of [3H]muscimol or [35S]t-butylbicyclophosphorothionate (TBPS) to forebrain homogenates, while pentobarbital was a less potent inhibitor of [35S]TBPS binding in the DBA strain. These findings suggest a genetic basis for the behavioral differences in convulsant sensitivity as well as for the neurochemical differences in allosteric coupling between convulsant and depressant/anticonvulsant sites associated with the GABA receptor-gated Cl- channel.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

35S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [35S]TBPS binding site on the GABAA receptor/Cl- ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS x SS RI) in addition to mice from the LS and SS lines. [35S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [35S]TBPS sites in SS mice was highest in the colliculi (4.5 +/- 0.5 pmol/mg protein), cerebellum (4.8 +/- 0.6 pmol/mg), hippocampus (3.2 +/- 0.7 pmol/mg) and cortex (2.9 +/- 0.3 pmol/mg). The Bmax was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in Bmax in any other area and in Kd values in any area (58 +/- 4.0 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress-induced behavioral depression in the rat is associated with a decrease in GABA receptor-mediated chloride ion flux and brain benzodiazepine receptor occupancy

Rats exposed to inescapable tailshock exhibit deficits in learning a simple shuttlebox escape task 24 h later. This syndrome has been termed 'behavioral depression' or 'learned helplessness', and is a model of stress-induced depression. In the present study a significant (25%) decrease in GABA receptor-mediated chloride ion flux as measured by muscimol-stimulated 36Cl- uptake in synaptoneurosomes was found in the cerebral cortices of rats that failed the shuttlebox task as compared to naive control rats. Rats which were exposed to tailshock and subsequently learned the escape task did not show a significant difference in muscimol-stimulated 36Cl- uptake as compared to naive control rats. Similarly, rats that failed to learn the shuttlebox escape task had significantly lower in vivo [3H]Ro15-1788 specific binding in cerebral cortex (43%), hippocampus (35%) and striatum (33%) as compared to naive control rats. In cerebellum and hypothalamus, there were significant reductions in specific [3H]Ro15-1788 binding in both animals that failed and animals that learned the shuttlebox escape task as compared to naive controls. To control the stress of the footshock associated with the shuttlebox escape task, we investigated the effect of gridshock in which total footshock received was equivalent to that received by rats who failed the shuttlebox task. There were no differences in muscimol-stimulated 36Cl- uptake or in vivo [3H]Ro15-1788 specific binding between naive controls and rats administered footshock independent of a learning task. These data suggest that the development of stress-induced behavioral depression may be associated with a decrease in GABA receptor-mediated chloride channel function.     

Dramatic increase in nigral t-[35S]butylbicyclophosphorothionate binding sites elicited by the degeneration of the striato-nigral GABAergic pathway: reversal by diazepam

In rats, the degeneration of the striato-nigral GABAergic pathway caused by the intrastriatal injection of kainic acid induced a marked decrease (65%) of GABA content and glutamic acid decarboxylase (GAD) activity and a dramatic increase (225%) in the binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a membrane preparation from the substantia nigra homolateral to the injected striatum. The increase in [35S]TBPS binding in the denervated substantia nigra was exclusively due to an increased density of binding sites (Bmax) with no change in the dissociation constant (kd). The enhancement in [35S]TBPS binding was almost completely reversed by the intraperitoneal administration of diazepam (3 mg/kg) to kainic acid-lesioned rats. Moreover, diazepam produced a significant decrease (30%) in the density of [35S]TBPS binding sites also in the sham-operated side. In contrast the 'in vitro' addition of the GABAA receptor antagonist bicuculline (1 microM) to the membrane preparation from the denervated substantia nigra further increased [35S]TBPS binding. These findings suggest the view that the increase of nigral [35S]TBPS binding is directly related to the inhibition in the function of nigral GABAergic synapses following the loss of the striato-nigral GABAergic pathway. Our results indicate that [35S]TBPS binding to brain structure is a potential tool to reveal alteration in the function of GABAA receptor complex elicited by physiological, pharmacological and pathological conditions.     

Etazolate (SQ20009): electrophysiology and effects on [3H]flunitrazepam binding in cultured cortical neurons

The actions of etazolate (SQ20009) on cultured cortical neurons have been examined in electrophysiological experiments and in receptor binding studies. Etazolate (0.3 to 100 microM) prolongs the duration of spontaneously occurring IPSPs. Higher concentrations of etazolate produce an increase in membrane chloride conductance, an effect which is picrotoxinin and bicuculline sensitive. Etazolate potentiates the response to exogenously applied GABA and acts synergistically with diazepam to enhance GABA-mediated conductance. Etazolate does not increase glycine-mediated conductance changes. Etazolate increases [3H]flunitrazepam binding and stimulates the GABA enhancement of [3H]flunitrazepam binding. In addition, GABA stimulates the etazolate enhancement of [3H]flunitrazepam binding. The etazolate-mediated increases in binding are Cl- dependent and picrotoxinin and bicuculline sensitive. The dose response relationships for etazolate-mediated effects are similar in physiological experiments and in binding studies. These data suggest that etazolate interacts with the postsynaptic GABA receptor complex at a site distinct from either the GABA recognition site or the benzodiazepine binding site to enhance GABA-mediated inhibition and to increase benzodiazepine binding.     

Aging: Effect on the interaction of ethanol and pentobarbital with the benzodiazepine-GABA receptor-ionophore complex

Benzodiazepine receptor binding and modulation by pentobarbital and ethanol was studied using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the gamma-aminobutyric acid (GABA)-benzodiazepine receptor-ionophore complex from the brains of Fischer 344 rats of 3-4, 12-15 and more than 28 months of age. The affinity of the benzodiazepine binding site was significantly lower in the young rats compared to either the mature or senescent animals. However, no age-related changes in the maximum number of benzodiazepine binding sites or GABA concentrations occurred in the detergent extract. Pentobarbital produced practically identical dose-dependent enhancement of [3H]flunitrazepam specific binding in all 3 age groups. In contrast, ethanol between 0.1 and 200 microM failed to produce a dose-dependent effect on [3H]flunitrazepam specific binding in any age group. The effect of pentobarbital and ethanol on [35S]t-butyl-bicyclophosphorothionate [( 35S]TBPS) specific binding to the picrotoxinin binding site was examined in the above solubilized receptor/ionophore complex under the same binding conditions. Both sedative-hypnotics produced a dose-dependent decrease in [35S]TBPS specific binding. However, pentobarbital was over 10,000 times more potent. It appears that ethanol may not enhance [3H]flunitrazepam specific binding in this solubilized preparation because of its weak action at the picrotoxinin binding site.     

[S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [³⁵S]TBPS binding site on the GABA_A receptor/Cl⁻ ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS × SS RI) in addition to mice from the LS and SS lines. [³⁵S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [³⁵S]TBPS sites in SS mice was highest in the colliculi (4.5 ± 0.5 pmol/mg protein), cerebellum (4.8 ± 0.6 pmol/mg), hippocampus (3.2 ± 0.7 pmol/mg) and cortex (2.9 ± 0.3 pmol/mg). The B_max was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in B_max in any other area and in K_d values in any area (58 ± 4.0 nM). When we compared [³⁵S]TBPS binding in the LS × SS RI strains, B_max values in IC were negatively correlated with latency to bicuculline-induced seizures but were not correlated with the duration of ethanol-induced sleeptime. These data support the hypothesis that differences in GABAergic neurochemistry in these mouse lines may not always be related to differences in ethanol sensitivity and point to GABAergic receptors in IC as possibly being involved in determining seizure sensitivity to bicuculline.     

Characterization of gamma-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions

Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.     

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABAA receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ ]flunitrazepam (2 nM), [ ]muscimol (5 nM), and 4 nM [ ]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ ]flunitrazepam and [ ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ ]flunitrazepam as well as [ ]muscimol binding (E_max) in the ethanol-dependent rat cerebellum as compared to control group (p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ ]TBPS at lower concentrations (100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ ]TBPS binding (−E_max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid_A (GABA_A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.     

Barbiturate tolerance: effects on GABA-operated chloride channel function

Male ICR mice were fed powdered laboratory chow containing phenobarbital for 7 days to induce tolerance. Mice were sacrificed and brains assayed for changes in GABA-mediated chloride flux into brain membrane vesicles (microsacs). Concentration-dependent stimulation of chloride flux by GABA alone was not affected by the development of tolerance to phenobarbital. Phenobarbital potentiation of GABA-mediated chloride flux was significantly attenuated in the membranes prepared from phenobarbital-tolerant mice compared with those from pair-fed control mice. Similarly, stimulation of GABA-mediated flux by the benzodiazepine, flunitrazepam was also depressed in membranes from tolerant mice. However, the ability of ethanol and the benzodiazepine inverse agonist FG-7142 to modulate GABA-gated chloride flux was not affected by the development of phenobarbital tolerance. No significant changes in saturation [3H]diazepam binding parameters were observed. These findings suggest that there is a degree of cross-tolerance between phenobarbital and benzodiazepine agonist at the level of the GABA-operated chloride channel. Furthermore, although some reports have demonstrated behavioral cross-tolerance between ethanol and barbiturates, the present data suggest different mechanisms of tolerance development for these intoxicants at the level of the GABAA receptor chloride channel complex.     

Interaction of a water-soluble derivative of delta-9-tetrahydrocannabinol with [3H]diazepam and [3H]flunitrazepam binding to rat brain membranes

We have tested the effect of a psychoactive water-soluble derivative of delta-9-tetrahydrocannabinol, SP-111A, on the binding of [3H]diazepam and [3H]flunitrazepam to rat brain membranes. It was found that SP-111A reduced the specific binding of [3H]diazepam and [3H]flunitrazepam. The inhibition by SP-111A was dependent not only on the concentration of the ligand but also on the protein content of membrane preparations. The inhibition of the specific binding of [3H]diazepam by SP-111A was found to be competitive with Ki value of 3.1 microM. In the presence of 7.5 microM SP111A the apparent Kd of [3H]diazepam binding increased from 4.3 nM to 12.5 nM, without affecting the Bmax. The inhibition of the specific binding of [3H]flunitrazepam by SP-111A was also competitive, however, the IC50 was higher than with [3H]diazepam. The inhibition by SP-111A appeared to be caused by its tight binding to the benzodiazepine binding sites of brain membranes.