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Benzodiazepine receptors were investigated in the cerebral cortex, the hippocampus, the brainstem, and the cerebellum of audiogenic seizure (AGS)-susceptible and seizure-resistant (ER) control rats. In AGS-susceptible rats of Sprague-Dawley descent, muscimol (10-6 M and 3 × 10-5 M) activated the binding of 3H-diazepam (0.4 nM) significantly less than in ER-rats. This finding may be strain selective, since it was not observed in AGS-susceptible rats of Wistar descent. Specific binding of the convulsant benzodiazepine receptor ligand methyl 6,7-dimethoxy-4-ethyl carboline-3-carboxylate (3H-DMCM), the benzodiazepine receptor ligand 3H-diazepam and the chloride channel directed cage convulsant t-butylbicyclophosphorothion-ate 35S-TBPS were not significantly changed in AGS-susceptible as compared to control rats. Our findings indicate that a disturbance at the level of the benzodiazepine receptor/GABA receptor/chloride channel complex is not a likely general aetiological factor for audigenic seizures in rats.

We have investigated the effect of unsaturated free fatty acids (FFAs) on the brain GABA/benzodiazepine receptor chloride channel complex from mammalian, avian, amphibian, and fish species in vitro. Unsaturated FFAs with a carbon chain length between 16 and 22 carbon atoms enhanced [3H]diazepam binding in rat brain membrane preparations, whereas the saturated analogues had no effect. The enhancement of [3H]diazepam binding by oleic acid was independent of the incubation temperature (0-30 degrees C) of the binding assay and not additive to the enhancement by high concentrations of Cl-. In rat brain preparations, the stimulation of [3H]diazepam binding by oleic acid (10(-4) M) was independent of the ontogenetic development. Phylogenetically, large differences were found in the effect of unsaturated FFAs on [3H]diazepam and [3H]muscimol binding: In mammals and amphibians, unsaturated FFAs enhanced both [3H]-muscimol and [3H]diazepam binding to 150-250% of control binding. In 17 fish species studied, oleic acid (10(-4) M) stimulation of [3H]diazepam binding was weak (11 species), absent (four species), or reversed to inhibition (two species), whereas stimulation of [3H]muscimol binding was of the same magnitude as in mammals and amphibians. In 10 bird species studied, only weak enhancement of [3H]muscimol binding (110-130% of control) by oleic acid (10(-4) M) was found, whereas [3H]diazepam binding enhancement was similar to values in mammal species. Radiation inactivation of the receptor complex in situ from frozen rat cortex showed that the functional target size for oleic acid to stimulate [3H]flunitrazepam binding has a molecular mass of approximately 200,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of compounds that open the GABAA receptor-chloride channel complex on the rapidly developed tolerance to the 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide(8-OH-DPAT)-induced hypothermia in rats were examined. The test compound was injected 15 min. before 1 mg/kg subcutaneous 8-OH-DPAT or saline and 24 hr later a challenge dose of 0.1 mg/kg subcutaneous 8-OH-DPAT was given. The rectal temperature was measured before the challenge dose and 30, 60, 90 and 120 min. thereafter. The hypothermic effect was calculated as the area under the curve. It was found that all the GABAergic compounds examined significantly counteracted the 8-OH-DPAT-induced tolerance to the hypothermic response: muscimol at 3 mg/kg subcutaneous, diazepam at 1 – 3 mg/kg subcutaneous, pentobarbitone sodium at 20 mg/kg subcutaneous, and chlormethiazole at 40 mg/kg subcutaneous. Combined treatment of the rats with the GABAA receptor antagonist, bicucculine, or the GABAA receptor-chloride channel blocker, picrotoxin and diazepam, pentoparbitone sodium or chlormethiazole significantly antagonised this counteraction of the 8-OH-DPAT-induced tolerance. Depletion of 5-HT by pretreatment of the rats with the tryptophan hydroxylase inhibitor p-chlorophenylalanine did not counteract the 8-OH-DPAT-induced tolerance to the hypothermic response. Pretreatment of the rats with dexamethazone did not change the development of the tolerance to 8-OH-DPAT-induced hypothermic effect which seems to exclude the involvement of the hypothalamo-pituitary-adrenocortical axis in the tolerance development. It is concluded that the results support the hypothesis that GABA neurones beyond the 5-HT neurones are involved in the mechanism causing tolerance to the 5-HT1A receptor-mediated hypothermia in rats.

THIS study examined the nature of the interactions of etifoxine, an anxiolytic and anticonvulsant compound, with the GABA A receptor/chloride channel complex. In membrane preparations of Sprague-Dawley rat cerebral cortex, etifoxine competitively inhibited the binding of [ 35 S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA A receptor chloride channel site. In vivo studies demonstrated an anticonvulsant effect of etifoxine (50 and 75 mg/kg, i.p.) against the clonic convulsions induced by TBPS in CD 1 mice. Flumazenil (10 and 40 mg/kg, i.p.), an antagonist of benzodiazepine sites at GABA A receptors, had no effect on the action of etifoxine. These findings suggest that etifoxine exerts its effect by interacting with the Cl - channel of GABA A receptors and probably by facilitating GABAergic inhibition.

1. General anesthetics at clinical concentrations are known to affect neurotransmitter-gated ion channels in postsynaptic membranes. 2. Volatile anesthetics suppress excitatory transmissions, whereas they potentiate inhibitory chloride currents evoked by GABA or glycine. Hexafluorodiethyl ether (a volatile convulsant) markedly depresses the GABA(A) response but not the glycine-evoked chloride current or the glutamate-induced excitatory response. 3. Molecular biology has revealed that GABA(A) receptor is a heteromeric pentamer composed of alpha, beta, gamma, delta, and/or rho subunits. In baculovirus-Sf9 expression system, the gamma subunit was crucial for potentiation of the GABA-induced chloride current by volatile anesthetics. 4. Following sustained presence of GABA and high concentrations of isoflurane, simultaneous washout of both agents evoked a slowly decaying surge current, whose nature is controversial.

1 Inhalational anaesthetics modulate ligand-gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for γ-aminobutyric acid (GABA) receptor modulation by isoflurane. 2 Wild-type Drosophila melanogaster homo-oligomeric GABA receptors were characterized and compared with an ion-channel mutant (alanine substituted to a serine in M2) by means of two-electrode voltage-clamp in membrane-invariant Xenopus oocytes. 3 Both channel receptor isoforms generated outwardly rectifying, bicuculline-insensitive currents with reversal potentials characteristic of a chloride current. 4 As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 μM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20. 5 Isoflurane, 195–389 μM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. 6 Isoflurane concentration-response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100–195 μM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 μM: wild-type receptors exhibited concentration-dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 μM). 7 The above divergence was not attributable to differential desensitization: neither wild-type nor mutant conductance desensitized significantly (P>0.05) in the absence or presence of anaesthetic. 8 This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. 9 The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. 10 The non-saturable enhancement of chloride conductances, when the PTX site is mutated, is not consistent with topical proposals that inhalational anaesthetics (stereoselectively) occupy a finite number of sites on these membrane spanning proteins. British Journal of Pharmacology (1997) 122, 726–732; doi:10.1038/sj.bjp.0701417

Changes in gamma-aminobutyric acidA (GABAA) receptor function and their relation to cross-tolerance to ethanol (EtOH) were studied in diazepam (DZP)-dependent rats. Physical dependence on DZP was induced in male Fischer rats by using the drug-admixed food method. The 38Cl- influx into cerebral cortical synaptoneurosomes induced by 10 microM GABA in DZP-withdrawn rats was significantly increased, compared with control and DZP-tolerant rats. Although enhancement of GABA-dependent 38Cl- influx by the addition of EtOH and flunitrazepam (FZ) was recognized in the control, there was no such effect of EtOH or FZ in the DZP-tolerant animals. On the other hand, GABA-dependent 38Cl- influx was enhanced by FZ in the withdrawn group. The addition of picrotoxin and bicuculline inhibited GABA-dependent 38Cl- influx in each group. The stimulatory effect of FZ on GABA-dependent 38Cl- influx was inhibited by the addition of Ro 15-1788 in the control group. However, such an inhibitory effect was not observed in the withdrawn group. The antagonistic effect of Ro 15-4513 on EtOH stimulation of GABA-dependent 38Cl- influx observed in the control was not recognized in the withdrawn group. In a [3H]FZ assay of binding to benzodiazepine (BZ) receptors, Bmax values were significantly increased in DZP-withdrawn animals, but decreased in the DZP-tolerant group, compared with the control. When [3H]muscimol binding was examined, the Kd of high-affinity sites of the GABAA receptor in withdrawn rats was significantly lower than in the control. In low-affinity binding sites, the values of Kd and Bmax were significantly decreased, compared with those in the control. The present study indicates that GABAergic transmission involving the regulation of GABA-dependent chloride channels is altered in DZP-dependent rats. Alterations of the GABAA/BZ/chloride channel complex function may be related to the cross-tolerance between BZ and EtOH.

The GABA A receptor-chloride channel complex is known to be the target of cyclodiene insecticides and of γ-hexachlorocyclohexane (γ-HCH, lindane). Dieldrin first enhances, then suppresses, γ-aminobutyric acid (GABA)- induced chloride currents, and the latter action results from the acceleration of desensitization. We now report more detailed analyses of the action of dieldrin and γ-HCH on the GABA A receptor-chloride channel complex of rat dorsal root ganglion, using whole-cell and single-channel patch clamp techniques. Preapplication of dieldrin suppressed GABA-induced chloride currents in a time and dose-dependent manner. No enhancement of current was observed under these conditions, because the enhancing action of dieldrin, which is transient in nature, had ceased by the time GABA-induced currents were recorded. Both transient and sustained components of chloride currents were suppressed by dieIdrin. The effect was almost irreversible, and the current did not recover after 30 min of washout with dieldrin-free solution. The currents activated by higher concentrations of GABA were more strongly suppressed by dieldrin. Coapplication of γ-HCH with GABA caused an enhancement of the GABA-induced chloride currents, which was followed by suppression. Single-channel currents induced by 1 μm GABA were completely suppressed by 1 μm γ-HCH. Hyperexcitation of the nervous system and whole animal caused by dieldrin and γ-HCH is the result of suppression of the GABA receptor-chloride channel complex

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

A voltage-gated chloride channel was identified in the electric organ of the marine ray Torpedo californica by White and Miller (J. Biol. Chem. 254, 10161–10166 (1979)). The experiments reported here concern the purification and identification of this channel which was accomplished by solubilization of electric organ plasma membranes and reconstitution of the channel into vesicles made of phosphatidylethanolamine, phosphatidylserine, and cholesterol. Channel activity was measured in these vesicles by assaying 36 Cl − uptake against an outwardly directed chloride chemical gradient as described by Garty et al. (J. Biol. Chem. 258, 13094–13099 (1983)). Maximal uptake occurred by 15 s. Addition of valinomycin after 10 min released intravesicular 36 Cl − suggestin that chloride is moving through a channel. Channel activity was inhibited by DIDS ( K 0.5 of 56 mM) and NBD chloride ( K 0.5 of 176 mM). In a 40 lipid/1 protein (w/w) reconstitution, approx. 30% of the vesicles contained a functional chloride channel, based upon uptake done in the presence of chlorotriphenyltin (an anion ionophore), indicating that the Torpedo electric organ is an enriched source as shown by White and Miller (Biophys. J. 35, 455–462 (1981)). The chloride channel was purified approx. 40-fold by sedimentation velocity. In this purified preparation, four polypeptides (210, 95, 55, and 40 kDa) were visible by silver-staining after nonreducing SDS-PAGE. Of the four polypeptides, the largest (210 kDa) is not sufficient for Cl − channel activity by itself, but it is labeled by DIDS, an inhibitor of channel activity. Channel activity was approx. 20-fold greater in material that bound to concanavalin A compared to the concanavalin A flow-through; all four polypeptides were present in the bound materia. It is possible that some of these polypeptides are subunits of the chloride channel.

The effect of chronic flurazepam treatment on the GABA (gamma-aminobutyric acid) receptor/chloride channel complex was studied using GABA-stimulated 36Cl- influx into brain microsacs, and its potentiation by diazepam, clonazepam and bretazenil. Rats were given flurazepam for 1 week, then microsacs were prepared from cerebral cortices of rats that were still receiving flurazepam, and from those that had stopped treatment 48 h earlier. Diazepam and clonazepam produced concentration-dependent increases in GABA-stimulated 36Cl- influx while bretazenil produced a much smaller effect, which did not reach statistical significance in the tissue from control rats. There was no significant change in the basal or 10 microM GABA-stimulated 36Cl- influx between control and treated groups. Tolerance was shown by a significantly reduced effect of diazepam and clonazepam to enhance GABA-stimulated 36Cl- influx in the tissue prepared from non-withdrawn rats. However, for both diazepam and clonazepam, there was no tolerance 48 h after chronic treatment. The results suggest that changes in the GABA receptor/Cl- channel complex on cerebral cortical neurons contribute to cross-tolerance from flurazepam to other benzodiazepines.

Sedatives and anesthetics can influence cerebral metabolism and respiratory and cardiovascular dynamics, which results in changes in cerebral perfusion. This is of major importance when functional brain imaging techniques are used to measure cerebral blood flow or to evaluate neurotransmitter systems, and also during neurosurgery. In the present review, the influences on brain perfusion of different sedatives including opioids and anesthetics commonly used in veterinary medicine are summarized.

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.

The regional distribution of radioactive ligand binding for different receptors of the gamma-aminobutyric acid A (GABAA)-benzodiazepine-picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from a genetic strain of Wistar rats with spontaneous absence-like seizures, the genetic absence epilepsy rats from Strasbourg (GAERS), and a control colony. The ligands employed included [3H]muscimol for high affinity GABA agonists sites; [3H]SR 95531 for the low-affinity GABA sites; [3H]flunitrazepam for the benzodiazepine sites; and [35S]t-butyl bicyclophosphorothionate (TBPS) for the picrotoxin site. There was no significant change between GAERS and control animals in [3H]flunitrazepam and [35S]TBPS binding. However, there was significantly decreased [3H]muscimol and [3H]SR 95531 binding in the CA2 region of the hippocampus of the GAERS. This was due to a decrease in Bmax of both [3H]muscimol and [3H]SR 95531 binding in the epileptic strain.

The effects of alprazolam (ALP), a triazolobenzodiazepine with high affinity for "central" benzodiazepine receptors, were examined on several parameters of immune function in mice. NK, MLR, and mitogen-induced lymphocyte proliferation were all significantly increased 2 hr after administration of low doses (0.02-1.0 mg/kg) of ALP. Twenty four hr later, similar but less robust immunoenhancing effects were observed. These measures of immune functions were not affected by higher doses of ALP (5-10 mg/kg). The immunoenhancing effects of ALP did not appear related to serum corticosterone levels. These and other findings demonstrate that the GABA/benzodiazepine receptor chloride channel complex can bidirectionally modulate immune function.

Agents that modify thiol groups have been shown to alter ligand binding at a variety of receptor sites. In addition, alkylation of sulfhydryls has been shown to block ion channel conductance. We studied the effects of thiol reagents on gamma-aminobutyric acid (GABA)-activated chloride flux (36Cl-) and [3H]-diazepam binding in mouse brain membrane preparation (microsacs). Incubation of microsacs in the presence of: mercuric chloride (HgCl2), p-chloromercuriphenylsulfonic acid (pCMBS), hydroxymercuribenzoate (HMB), n-ethylmaleimide (NEM), or iodoacetic acid (IAA) attenuated GABA-stimulated Cl- uptake. The thiol reagents reduced both maximal stimulation and the potency of GABA to induce Cl- uptake. Thiol reagent treatment decreased the affinity of high-affinity [3H]-muscimol equilibrium binding. Supernatant prepared from microsacs treated with pCMBS stimulated Cl- uptake in the absence of GABA agonist in microsacs unexposed to thiol reagents. The supernatant taken from pCMBS-treated microsacs also stimulated [3H]-diazepam binding. This effect was blocked by the addition of the GABA receptor antagonist bicuculline. The concentration of endogenous GABA in supernatant from pCMBS-treated microsacs was sixfold greater than that in supernatant from control microsacs. This increase in levels of endogenous GABA by thiol reagents was due to both an increase in GABA release and a decrease in high-affinity GABA uptake.

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

Summary Considerable evidence suggests that at least some of the effects of ethanol are mediated by an action on the GABAA receptor chloride channel complex. More speculative is the suggestion that ethanol might interact with endogenous ligands for the benzodiazepine receptor on the complex. This paper considers the evidence for such interactions.

The basic defect in cystic fibrosis is the chloride impermeability of the plasmalemm in different cells. A candidate for the chloride channel, thought to be affected in the syndrome, is “Porin 31HL” recently described by us. The molecule is i) expressed in the plasmalemm of different cells, it has ii) a molecular mass of 31000 Daltons, it shows iii) high conductance in artificial membranes and it can be iv) modified by 4,4′-Diisothiocyana-tostilben-2,2′-disulfonat. A porin in the outer membrane of cells should furthermore v) be regulated by modulators. All these characters of “Porin 31HL” correspond to those given in literature for chloride channels. The regulation of the channels can be explained by a two component flip flop model.

The effects of gamma-aminobutyric acid (GABA) and related agonists and antagonists on the excitatory cell death were examined in dispersed primary cultures of the rat cerebral cortex. The cytotoxic effects evoked by kainic acid, quisqualic acid and N-methyl-D-aspartic acid were evaluated by phase contrast microscopy and quantified by the measurement of lactic dehydrogenase release into the culture medium. GABA accelerated the cell death in a concentration-dependent fashion, but did not influence the amount of cells dying with 24 h after the treatment. This effect of GABA could be mimicked by GABAA rather than GABAB receptor agonists. Blockers acting at different sites of the GABAA receptor/chloride channel complex not only reduced the GABA-induced acceleration of cell death, but also showed a significant protection against the excitotoxic cell death in the absence of exogenous GABA. Blockers of chloride channels unrelated to GABA receptors produced similar protection. Our findings indicate that GABA-gated chloride channels may be effective in modulating excitotoxic vulnerability of cerebrocortical cells.

7 pages, 6 figures, 1 table.-- Available online Dec 23, 2007., The silphinenes are tricyclic sesquiterpenes that have antifeedant and toxic effects in insects and structural similarity to the known GABA antagonist, picrotoxinin. In murine synaptoneurosomes, silphinenes block GABA-stimulated influx of Cl-36(−) with EC-50s in the range of 10–30 μM. In insects, silphinenes were tested in neurophysiological recordings of central neurons from third instar Drosophila melanogaster larvae. Silphinenes reversed the blockage of neuronal firing induced by GABA, but had little effect below 100 μM. The structure–activity profile observed in the murine chloride flux assay was also observed in the larval neurophysiological assay, indicating little selectivity for the silphinenes. A reference silphinene was equally active on nerve preparations from the rdl strain of D. melanogaster, which is resistant to channel-blocking antagonists via an altered GABA receptor. This latter finding suggests that silphinenes interact with the insect GABA receptor in a manner somewhat different from PTX, and that rdl resistance in the field may have little effect on silphinene efficacy., We thank the Virginia Tech Biological Sciences Initiative for funding this research, as well as Grant CTQ2006-15597-C02-01/PPQ from the government of Spain.

A model for the development of pentobarbital tolerance and dependence was characterized and correlated with changes in radioligand binding to the GABAA-benzodiazepine receptor chloride channel complex. While one day of pentobarbital exposure decreased the duration of loss of righting reflex, tolerance to the hypothermic effects of thiopental and barbital took 7 days to develop, indicating that pharmacokinetic and pharmacodynamic tolerance are separable. Increased sensitivity to pentylenetetrazol-induced seizures was first observed after 3 days of pentobarbital exposure, suggesting brain areas involved in seizure control develop tolerance to, and dependence on pentobarbital faster than those involved in temperature regulation. Acute exposure to pentobarbital in vivo did not affect cortical binding of [3H]muscimol in vitro, while tolerance caused a decrease in binding due to an increase in the low-affinity site KD. Pentobarbital tolerance also caused a decrease in the cortical binding of the benzodiazepine, [3H]flunitrazepam. These observations suggest that the acute effects of barbiturates on the GABAA receptor complex are reversible, while tolerance causes receptor modifications which may be related to the development of physical dependence.

The effects of gamma-aminobutyric acid (GABA) on the glutamate receptor chloride ion (Cl-) channel complex were examined in mechanically isolated and internally perfused Aplysia neurons using a concentration clamp technique. GABA at concentrations of 3 x 10(-6) M or more, concentration dependently delayed the recovery of the glutamate response from desensitization. This effect was independent of the GABA response and Cl- redistribution. Muscimol (10(-4) M) mimicked the effect of GABA. However, this was not the case for baclofen (10(-3) M). In some isolated neurons, GABA at concentrations of more than 10(-4) M clearly induced an additional Cl- current, the current kinetics of which were different from those induced by lower concentrations of GABA. Even in the continued presence of 10(-4) M GABA, which desensitized the fast GABA response, higher concentrations of GABA (3 x 10(-4) M to 10(-2) M) elicited the additional current in a concentration-dependent manner. The presence of 10(-4) M glutamate completely abolished this current, indicating cross-desensitization between the glutamate and slow GABA responses. High concentrations of GABA (3 x 10(-2) M) did not activate the glutamate receptor coupled to the large cation channel. The results suggest that, in Aplysia neurons, the glutamate receptor-Cl- channel complex has some similarities to the GABA receptor-Cl- channel complex.

Synthesis of octyl-O-beta-D-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6-rerra-O-benzyl-1-O-1',3'2'-dioxaphosphacyclohexane-a lpha,beta-D-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl triflate) and subsequent debenzylation. At 100 microM the molecule significantly and reversibly increased the magnitude of GABA(A) currents evoked in cultured rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier proposals that such molecules bind to the TBPS (tert-Butylbicyclophosphorothionate) site to block the chloride channel.

Steroids with the 3alpha-hydroxy-5alpha- or 5beta-reduced configurations of the A ring interact with the gamma-aminobutyric acid (GABA) type A receptor chloride channel complex and potentiate the stimulation of Cl- uptake by GABA agonists. Conversely, the sulfate esters of 3beta-hydroxy-5-ene neurosteroids pregnenolone and dehydroepiandrosterone behave as inhibitory modulators. In the present work, steroid sulfates were tested for their ability to modulate muscimol-induced chloride ion uptake into cortical synaptoneurosomes. 3alpha-Hydroxy-5alpha-pregnan-20-one sulfate and several other 3alpha-hydroxy-steroid sulfates potentiated, whereas 3beta-hydroxy-steroid sulfates inhibited muscimol effect. It is concluded that GABA-agonistic or antagonistic properties of steroid sulfates depend on the alpha or beta orientation of the sulfate moiety linked to the A ring.

Certain types of neuronal ions channels have been demonstrated to be the major target sites of insecticides. The insecticide-channel interactions that have been studied most extensively are pyrethroid actions on the voltage-gated sodium channel and cyclodiene/lindane actions on the GABAA receptor chloride channel complex. With the exception of organophosphate and carbamate insecticides which inhibit acetylcholinesterases, most insecticide commercially developed act on the sodium channel and the GABA system. Pyrethroids show the kinetics of both activation and inactivation gates of sodium channels resulting in prolonged openings of individual channels. This causes membrane depolarization, repetitive discharges and synaptic disturbances leading to hyperexcitatory symptoms of poisoning in animals. Only a very small fraction (approximately 1%) of sodium channel population is required to be modified by pyrethroids to produce severe hyperexcitatory symptoms. This toxicity amplification theory applies to pharmacological and toxicological action of other drugs that go through a threshold phenomenon. Selective toxicity of pyrethroids between invertebrates and mammals can be explained based largely on the responses of sodium channels and partly on metabolic degradation. The pyrethroid-sodium channel interaction is also supported by Na+ uptake and batrachotoxin binding experiments. Cyclodienes and lindane exert a dual action on the GABAA system, the initial transient stimulation being followed by a suppression. The stimulation requires the presence of the gamma 2 subunit. The suppression of the GABA system is also documented by Cl- flux and ligand binding experiments. It appears that the sodium channel and the GABA system merit continuing efforts for development of newer and better insecticides. Nitromethylene heterocycles including imidacloprid act on nicotinic acetylcholine receptors. Insect receptors are more sensitive to these compounds than mammalian receptors. Single-channel analyses of the nicotinic acetylcholine receptor of PC12 cells have shown that imidacloprid increases the activity of subconductance state currents and decreases that of main conductance state currents. This may explain the imidacloprid suppression of acetylcholine responses.

The effects of n-alcohols on the γ -aminobutyric acid (GABA) receptor-Cl− channel complex of rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch-clamp technique. n-Alcohols (C1 – C11) enhanced the GABA-induced current, their effects, increasing with their carbon chain lengths. n-Alcohols (C2, C8) accelerated desensitization of the GABA-induced current. At higher concentrations, n-alcohols (C2, C4, C6 and C8) alone gated the Cl− channel of the receptor-channel complex to generate a Cl− current.

Barbiturates are central nervous system depressants that are used as sedatives, hypnotics, anesthetics and anticonvulsants. However, prolonged use of the drugs produces physical dependence, and the drugs have a high abuse liability. The gamma-aminobutyric acidA (GABAA) receptor is one of barbiturates' main sites of action, and therefore it is thought to play a pivotal role in the development of tolerance to and dependence on barbiturates. Recent advances in the study of the GABAA receptor/chloride channel complex allow us to examine possible mechanisms that underlie barbiturate tolerance/dependence in a new light. In this minireview, we mainly focus on molecular and cellular aspects of the action of barbiturates and the possible mechanisms that contribute to development of tolerance to and dependence on barbiturates.

The in vitro receptor-stimulated synaptoneurosomal chloride uptake induced by pentobarbital or muscimol was studied in the brains of rats which had been rendered tolerant to or dependent on pentobarbital. In the chronic study, rats received continuous administration of sodium pentobarbital by intracerebroventricular (i.c.v.) infusion. In rats continuously infused with pentobarbital for 6 days or in rats which had the infusion terminated for 24 h, the basal synaptoneurosomal chloride uptake was not altered in either the cerebral cortex or the cerebellum. On the other hand, pentobarbital (500mgr;M)-stimulated chloride uptake was significantly decreased in both cerebral cortices and cerebella of rats which received pentobarbital for 6 days as compared with the saline control groups. Twenty-four hours after termination of pentobarbital infusion, this high concentration of pentobarbital-stimulated chloride uptake remained low in both cerebral cortices and cerebella of rats which had been infused with pentobarbital for 6 days. In addition, similar results were also obtained in muscimol (2.5mgr;M)-stimulated chloride uptake in the cerebral cortices of pentobarbital-infused animals. However, pentobarbital infusion failed to alter muscimol-stimulated chloride uptake in the cerebellum. These results suggest that the GABA(A) receptor regulated chloride uptake is downregulated after chronic pentobarbital administration. The results also suggest that down-regulation of the GABA(A) receptor chloride channel complex takes place at different recognition sites on the complex. It further substantiates the allosteric effects of GABA and barbiturate recognition sites. Copyright 1996 S. Karger AG, Basel

The GABAA receptor-chloride channel complex has recently been demonstrated by patch clamp experiments to be the target of cyclodiene insecticides. We have now examined the effects of four isomers of hexachlorocyclohexane (HCH), alpha-, beta-, gamma- and delta-HCH, on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons using patch clamp techniques. When co-applied with 10 microM GABA, 1 microM gamma-HCH slightly enhanced and then suppressed the GABA-induced chloride current. The desensitization of the current was greatly accelerated by gamma-HCH in a dose-dependent manner. The acceleration of desensitization and the suppression of sustained component of current by gamma-HCH occurred at lower concentration ranges than those for the suppression of peak current. When 10 microM delta-HCH was co-applied with 10 microM GABA, current was greatly enhanced and then suppressed, and the level of enhancement was much higher than that of gamma-HCH. alpha- and beta-HCH had little or no effect on the GABA-induced chloride current. The differential actions of these isomers on GABA-activated chloride currents account for variable symptoms of poisoning in insects and mammals.

A surge of pituitary luteinizing hormone (LH) into the bloodstream occurs in hamsters every 4 days between 1:30 p.m. and 3 p.m. in response to a signal from a biological clock. This surge initiates behavioral estrus approximately 2 h later and ovulation approximately 12 h later. Phenobarbital at a doseor = 100 mg/kg consistently blocks LH release. Barbiturate and benzodiazepine drugs have separate binding sites in the GABAA receptor/chloride channel complex. Binding of either drug increases GABA-mediated chloride conductance, which suppresses the postsynaptic neuron. Barbiturate binding also increases benzodiazepine binding. This suggested that these drugs might synergize to inhibit LH release. A combination of triazolam and phenobarbital at doses of 10 mg/kg injected s.c. at 1:30 p.m. inhibited ovulation and extended the 4-day vaginal cycle in all treated hamsters. Either drug dose injected alone at 1:30 p.m., or the combination at 3 p.m., was completely ineffective. Bicuculline prevented inhibition by the combination at 1:30 p.m. The clock signal for LH release may act by antagonizing GABA transmission, which may be chronically inhibiting LH release. The combination delimited a 75-min period (1:30-2:45 p.m.) within which the clock signal for LH release occurred in all individuals (ET50 = 2:08 p.m.). This period appears to arise from individuals with different but constant clock settings rather than from a 75-min variation in the clock setting of the individual.

GABAA-receptors (GABARs) are chloride ion channels in the ‘cys-loop receptor’ superfamily that are major inhibitory neuroreceptors. Upon agonist binding, GABARs open and increase intraneuronal chloride ion concentration, hyperpolarize the cell and inhibit neural transmission. Reducing agents will break the disulfide bonds in the cys-loop of the receptor. Indeed, it has been speculated that sulfhydryl compounds modify proteins via thiol/disulfide redox reactions and serve as neuromodulators (1). Reducing reagents were found to decrease the GABA EC50 in the highly homologous GABAC-receptor, while oxidizing reagents increased the EC50 (2). Conversely, conflicting findings suggests the mechanism of action of redox modulation does not alter GABAR agonist-binding affinity (3). However, studies are complicated by the fact that mutagenesis of the cysteines commonly produces failure of subunit assembly (4). Thus, to investigate the effect of disulfide bond breakage on the receptor we generated various GABAR homology models with and without disulfide bonds. These systems were subjected to ligand docking and extensive molecular dynamics simulations to determine if the GABA affinity was modulated. This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.1. Allan, A. M. and L. D. Baier. 1992. Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex. Synapse 10:310-316.2. Calero, C. I. and D. J. Calvo. 2008. Redox modulation of homomeric rho1 GABA receptors. J Neurochem 105:2367-2374.3. Pan, Z. H., et al. 2000. Redox modulation of recombinant human GABA(A) receptors. Neuroscience 98:333-338.4. Amin, J., et al. 1994. The agonist binding site of the gamma-aminobutyric acid type A channel is not formed by the extracellular cysteine loop. Mol Pharmacol 45:317-323.LLNL-ABS-502871

An analogue of colchicine, beta-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. beta-Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol-stimulated 36Cl- uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopus oocytes expressing human alpha 1 beta 2 gamma 2S, but not alpha 1 beta 2, GABAA receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of beta-lumicolchicine on the GABAA receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, beta-lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-THP) acts as a potent allosteric modulator and a direct activator of the GABA-chloride channel complex. This neurosteroid has also been found to protect against seizures that arise from blockade of the GABA- chloride channel complex. Because 3 alpha,5 alpha-THP protects against excitotoxin-induced seizure activity and because seizure activity has been found to be associated with aberrant hippocampal nerve cell growth, the rapid effect of the neurosteroid 3 alpha,5 alpha-THP upon nerve cell growth was investigated using videomicroscopy of hippocampal neurons in culture. Within 40 min of exposure 3 alpha,5 alpha-THP induced a significant decrease in the area and length of neurites. A concomitant decrement in the number and length of filopodia decorating neuritic extensions also occurred within the 40 min of 3 alpha,5 alpha- THP exposure. Both rapid and slow retrograde movement of intracellular organelles was observed in 3 alpha,5 alpha-THP-treated neurons. 3 alpha,5 alpha-THP-induced regression of neuritic extensions occurred only in nerve cells that had not yet established contact with other nerve or glial cells in culture. Established structural connections between neurons or glia did not erode during 3 alpha,5 alpha-THP exposure. Neither the inactive stereoisomer 3 beta-hydroxy-5 beta- pregnan-20-one nor progesterone had a significant effect upon any of the morphological parameters assessed. In approximately 25% of the cells in which 3 alpha,5 alpha-THP had induced regression, subsequent exposure to 17 beta-estradiol induced profuse filopodial growth within 60 sec of exposure. In cultures similar in age to those used in the morphological studies, 3 alpha,5 alpha-THP induced a significant increase in 36Cl- uptake within 10 sec. The magnitude of 36Cl- uptake was comparable to that induced by exposure to 100 microM GABA. In older, more mature cultures in which the nerve cells had established structural connections, 3 alpha,5 alpha-THP protected cells from picrotoxin-induced nerve cell death. These results demonstrate that 3 alpha,5 alpha-THP can induce regression of neuronal morphology within a relatively rapid time frame. 3 alpha,5 alpha-THP induction of 36Cl- uptake within 10 sec suggests that activation of neurosteroid-regulated chloride channels is an initial step in the biochemical mechanism underlying the retraction induced by this progesterone metabolite steroid. In select instances, 17 beta-estradiol induced an extremely rapid reversal of the filopodial regression produced by 3 alpha,5 alpha- THP.(ABSTRACT TRUNCATED AT 400 WORDS)

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

In 1989, showing the expression of “Porin 31HL” in the plasmalemma of human B lymphocytes, our laboratory gave first evidence on the multitopological localization of VDAC. Looking for a function of the channel in the outer cell membrane, we referred to five corresponding traits shared by “Porin 31HL” and plasmalemma integrated mammalian chloride channels. We furthermore proposed VDAC to be part of a chloride channel complex. VDAC on outer cell surfaces needs biochemical regulation. In apical membranes of epithelia it might be regulated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Our model on the interaction of VDAC and CFTR may reconcile data on the outwardly rectifying depolarization-induced chloride channel (ORDIC) and the ohmic low-conductance channel formed by the CFTR. Both channels are affected in Cystic Fibrosis and very recent data support that CFTR might regulate ORDIC. As basis for immunotopological work our laboratory had purified and sequenced “Porin 31HL” on the protein level, thus giving the first primary structure analysis of a VDAC from the animal kingdom. Comparing the topochemistry, the channel characteristics and the primary structure of VDAC from normal and Cystic Fibrosis B-lymphocyte cell lines, we were looking for indications of the involvement of VDAC in the CF syndrome.

This study investigated the pharmacological profile of cyclodiene resistance in Drosophila melanogaster and the mode of action of a phenylpyrazole insecticide, JKU 0422. Toxicological studies were performed with a sucrose bait assay containing the synergist piperonyl butoxide. The Maryland strain of D. melanogaster was resistant to dieldrin, lindane, picrotoxinin, TBPS, p-CN-TBOB, and JKU 0422. In contrast, this strain was susceptible to cypermethrin and the avermectins MK-243, abamectin, and abamectin 8,9-oxide. Neurophysiological studies showed that both TBPS and JKU 0422 reversed the inhibitory action of GABA in central nerve preparations from susceptible D. melanogaster. However, the response to these compounds was attenuated in nerve preparations from the resistant Maryland strain, which indicated that the resistance was expressed at the level of the nerve. Topical toxicity bioassays with JKU 0422 on susceptible (CSMA) and cyclodiene-resistant (LPP) strains of German cockroach revealed a resistance ratio of 553-fold for this compound. These studies demonstrate that cyclodiene resistance in D. melanogaster confers broad cross resistance toward compounds thought to block the GABA-gated chloride channel in a manner similar to the cyclodienes. Moreover, the cross resistance extends to JKU 0422, and resistance to this compound is also present in a strain of cyclodiene-resistant German cockroach. These toxicological results, along with the neurophysiological studies, confirm that JKU 0422 has a mode of action that is similar to the cyclodienes and TBPS. These findings suggest that the introduction and use of new chloride channel antagonists as insecticides should be managed carefully in order to prevent the rapid development of resistance in the field.

Publisher Summary One of the major neurotransmitters in the mammalian central nervous system is γ-aminobutyric acid (GABA). This chapter presents methods to study GABAA receptor-mediated Cl- responses and discusses some aspects of the modulation by extracellular agents. The GABAA receptor belongs to a ligand-gated receptor superfamily with a typical four-transmembrane-spanning motif. There are three types of GABA receptors: (1) GABAA receptors, which are bicuculline-sensitive and directly coupled with an integral Cl- channel; (2) GABAB receptors, which are indirectly coupled with K+ and/or Ca2+ channels through GTP-binding proteins; and (3) GABAC receptors, which operate bicuculline-insensitive Cl- channels. GABAA receptors have numerous sites of modulation and structural diversity inferred from molecular biology. They contain binding sites for therapeutically significant drugs, including convulsant, hypnotic–anticonvulsant, anxiolytic, and anxiogenic agents. Endogenous agents such as steroids and intracellular Ca2+, ATP, and cyclic AMP modulate the GABAA receptor. The number and mode of actions give rise to a level of complexity comparable only to that of N-methyl- d -aspartate receptors.

1. The effect of various classes of compounds on the rapidly developed tolerance to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-induced corticosterone secretion was examined. 2. Compounds activating the gamma-aminobutyric acidA (GABAA) receptor-chloride complex, i.e. muscimol (3 mg kg-1), diazepam (5 mg kg-1), flunitrazepam (1 mg kg-1), sodium pentobarbitone (10-30 mg kg-1) and chlormethiazole ethane disulphonate (50 mg kg-1) counteracted the development of tolerance when injected before or simultaneously with, but not 15 min after 8-OH-DPAT. 3. At these doses the compounds produced an acute increase in serum corticosterone but had, with the exception of muscimol, no effect on the response to the challenge dose of 8-OH-DPAT 24 h later. Muscimol significantly decreased the response. 4. The GABAA chloride channel antagonist, picrotoxin (1 mg kg-1, s.c.), but not bicuculline (1 mg kg-1, i.p.) potentiated the development of tolerance to 8-OH-DPAT-induced corticosterone secretion. 5. A number of compounds with widely differing pharmacological actions were examined and found to have no effect on the development of tolerance to 8-OH-DPAT-induced corticosterone secretion.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.

The presence of GABA A receptors (GABARs) in auditory brainstem nuclei of the chick was determined by immunocytochemical (ICC) and receptor autoradiographic techniques. A monoclonal antibody to the GABAR/benzodiazepine/chloride channel complex and radiolabeled ligand binding using [ 3 H]-muscimol, a GABA agonist, revealed labeling in nucleus magnocellularis (NM), nucleus laminaris (NL), nucleus angularis (NA), and the superior olive (SO) in both posthatch and embryonic chicks. GABAR-immunoreactivity (GABAR-I), as well as [ 3 H]-muscimol binding, appear homogeneous throughout these nuclei at all ages studied. During development, GABAR-I is first observed in these nuclei around embryonic day 13 (E13). GABAR-I, which appears heavier in embryos than in posthatch chicks, becomes less intense with age in all 4 nuclei. Levels of receptor binding are also greater in embryos compared to posthatch chicks. [ 3 H]-Muscimol binding is consistently greatest in SO followed by that in NL. NM and NA exhibit the least amount of binding at all ages studied. [ 3 H]-Muscimol binding decreases in auditory brainstem nuclei as a function of age. Two days after unilateral cochlea removal, there is an apparent increase in GABAR-I in the ipsilateral NM compared to controls. This, however, may be the result of a decrease in the cross-sectional area of NM neurons as a result of de-afferentation (Born and Rubel, 1985). In contrast, there is a 28% decrease in [ 3 H]-muscimol binding in the ipsilateral NM compared to controls probably reflecting the 30% reduction in the number of NM neurons due to cochlea removal (Born and Rubel, 1985). Fourteen days after cochlea removal, there is still a small, but not significant, decrease in [ 3 H]-muscimol binding in the ipsilateral NM. In the contralateral NM, GABAR-I is less intense compared to that in the ipsilateral NM and controls. Additionally, there is a slight but insignificant decrease in [ 3 H]-muscimol binding compared to that in controls 2 days after cochlea removal. After 14 days survival, however, the average binding is similar to that in controls. Thus, cochlea removal appears to transiently decrease the number of GABARs in the ipsilateral NM and may have a similar, but not as dramatic, effect in the contralateral NM. These GABARs are most likely to be postsynaptic, that is, located on NM neurons.

Function of the benzodiazepine (BZD)/GABA receptor chloride channel complex was selectively altered by specific aspects of an environmental challenge, i.e. encountering a stranger in a familiar versus an unfamiliar environment. Chloride (Cl-) enhancement of [3H]flunitrazepam [( 3H]flu) binding was facilitated in rats tested in an unfamiliar environment relative to that in rats tested in a familiar environment. Basal [3H]flu binding (binding in the absence of NaCl) also was greater in rats tested in the unfamiliar environment than in rats tested in the familiar environment, and Scatchard analysis of [3H]flu binding indicated that increased [3H]flu binding in the unfamiliar environment reflected an increase in both binding affinity and maximal binding capacity. In addition, both the sensitivity of [3H]flu binding to Cl- and the affinity of BZD recognition sites were decreased in handled control rats relative to non-handled control rats as well as to environmentally-challenged (prehandled) rats, suggesting that the experience of daily handling as well as familiarization with the environment modulates function at the BZD/GABA receptor complex. GABA-mediated 36Cl- uptake was facilitated by testing in either the familiar or unfamiliar environment relative to that measured in non-handled control rats. Thus, changes in GABA-gated chloride channel function may reflect a more fundamental response of this complex to challenging situations. These findings suggest that components of the BZD/GABA receptor complex are differentially influenced by specific aspects of an environmental challenge. Furthermore, function at the BZD recognition site/chloride channel component of this receptor complex is influenced by both repeated and single exposure to specific environments.

A dose-dependent (12.5–100 mg/kg) suppression of cytotoxic T lymphocyte (CTL) activity was observed in mice after administration of the benzodiazepine receptor ‘inverse agonist’ FG 7142 (N-methyl-β-carboline-3-carboxamide). This compound produces a syndrome resembling stress or anxiety in both animals and man. Addition of FG 7142 (1–1000 nM) to either a 4-hour 51Chromium-release assay or 5-day in vitro CTL generation system did not affect CTL activity. Pretreatment with the benzodiazepine receptor antagonist Ro 15-1788 (10 mg/kg) attenuated FG 7142-induced suppression of CTL activity, but had no effect when administered alone. Time-course studies indicated that FG 7142-induced suppression of CTL activity was long-lasting. The suppression of CTL activity by FG 7142 provides further evidence that the central nervous system pathways subserved by the benzodiazepine/GABA receptor chloride channel complex may play an important role in the modulation of immune function.

The distribution of the GABAA receptor/benzodiazepine receptor/chloride channel complex was investigated in the thalamus of the rat by means of immunohistochemistry in adulthood, as well as during embryonic and postnatal development, using a monoclonal antibody. In adults, the immunoreactivity for the GABAA receptor complex was intensely expressed by neuronal processes throughout the dorsal thalamus. Neuronal perikaryal membranes were frequently outlined by punctate immunostaining; cell bodies, intrathalamic fibre bundles and the internal capsule did not display immunoreactivity for the GABAA receptor. Regional differences in the expression of the receptor were consistently observed: the immunostaining was much lighter in the thalamic reticular nucleus than in the dorsal thalamic nuclei and, among the latter, the anteroventral nucleus and the ventral nuclear complex displayed the most intense immunopositivity. Immunostaining for the GABAA receptor was already expressed in embryos at E14, and was homogeneously distributed throughout the neuropil of the dorsal and ventral thalamic primordia. During the first two postnatal weeks, a regional differentiation of the immunopositivity was appreciable in the thalamus, with a progressive reduction in the reticular nucleus and a parallel increase in the dorsal thalamic structures. Immunoreactive neuronal perikarya were not observed in the thalamus at any developmental stage. The expression of the GABAA receptor complex appeared to have reached a mature configuration by the end of the third postnatal week. These findings indicate that in adults the GABAA receptor is differentially expressed by thalamic nuclear structures, including the reticular nucleus. Furthermore, the maturation of the receptor in the thalamus undergoes a rearrangement during the first postnatal weeks that results in a considerable regression within the reticular nucleus.