Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.

1. The modulation of the gamma-aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage-clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]-muscimol to a preparation of porcine brain membranes. 2. The progesterone metabolites 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one (greater than or equal to 30 nM) reversibly and dose-dependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 microM), and over the concentration range 30 nM-100 microM stimulated the binding of [3H]-muscimol. In contrast, 5 alpha- and 5 beta-pregnan-3 beta-ol-20-one (30 nM-100 microM) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3. Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]-muscimol binding sites as the principle action of the active steroid isomers. 4. GABA-evoked currents were also potentiated by androsterone (1 microM) and the deoxycorticosterone metabolite 5 alpha-pregnane-3 alpha,21-diol-20-one (100 nM). 5. Secobarbitone (10-100 microM), pentobarbitone (10-300 microM) and phenobarbitone (100-500 microM) reversibly and dose-dependently potentiated the amplitude of GABA-evoked currents in the absence of any change in their reversal potential. 6. At relatively high concentrations (greater than or equal to 30 microM) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor-channel activation since they share a common reversal potential with GABA-evoked responses (approximately 0 mV), are reversibly antagonized by bicuculline (3 microM), and potentiated by either diazepam (1 microM) or 5 beta-pregnan-3 alpha-ol-20-one (500 nM). 7. Secobarbitone (1 microM-1 mM) dose-dependently enhanced the binding of [3H]-muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8. A saturating concentration (1 mM) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5 beta-pregnan-3 alpha-ol-20-one (30 nM-100 microM). Similarly the steroid, at a concentration of 100 microM, did not influence the enhancement of [3H]-muscimol binding by secobarbitone (1 microM-1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones.

1. The interaction of the intravenous general anaesthetic propofol (2,6-diisopropylphenol) with the GABAA receptor has been investigated in voltage-clamped bovine chromaffin cells and rat cortical neurones in cell culture. Additionally, the effects of propofol on the glycine and GABAA receptors of murine spinal neurones were determined. 2. Propofol (1.7-16.8 microM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by GABA (100 microM) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 microM) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4 microM and 1.7-16.8 microM propofol respectively produced a potentiation of GABA-evoked currents qualitatively similar to that seen in the bovine chromaffin cell. 3. The potentiation by propofol (1.7 microM) was not associated with a change in the reversal potential of the GABA-evoked whole cell current. On outside-out membrane patches isolated from bovine chromaffin cells, propofol (1.7 microM) had little or no effect on the GABA single channel conductances, but greatly increased the probability of the GABA-gated channel being in the conducting state. 4. The potentiation of GABA-evoked whole cell currents by propofol (1.7 microM) was not influenced by the benzodiazepine antagonist flumazenil (0.3 microM). A concentration of propofol (1.7 microM) that substantially potentiated GABA currents had little effect on currents induced by the activation of the GABAA receptor by pentobarbitone (1 mM). 5. Bath application of propofol (8.4-252 microM), to bovine chromaffin cells voltage clamped at -60 mV, induced an inward current associated with an increase in membrane current noise on all cells sensitive to GABA. Intracellular application of propofol (16.8 microM) was ineffective in this respect. Local application of propofol (600 microM) induced whole cell currents with a reversal potential dependent upon the Cl- gradient across the cell membrane. 6. On outside-out membrane patches formed from bovine chromaffin cells, propofol (30 microM) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 microM bicuculline. Under identical ionic conditions, GABA (1 microM) activated single channels with mean chord conductances of 33, 16 and 10pS. 7. Bath applied propofol (0.84-16.8 microM) dose-dependently potentiated strychnine-sensitive currents evoked by glycine (100 microM) in murine spinal neurones. 8. The relevance of the present results to the general anaesthetic action of propofol is discussed.     

Potentiation, activation and blockade of GABAA receptors of clonal murine hypothalamic GT1-7 neurones by propofol.

1. The actions of GABA and the intravenous general anaesthetic propofol (2,6-diisopropylphenol) on GABAA receptors of self-replicating GT1-7 hypothalamic neurones were investigated by the patch clamp technique. 2. GABA (1 microM-1 mM) dose-dependently activated inward currents with an EC50 of 27 microM, recorded from whole cells voltage-clamped at -60 mV. GABA (100 microM)-activated currents reversed at the Cl-equilibrium potential. 3. Propofol (0.1-100 microM) dose-dependently potentiated GABA (100 microM)-evoked currents with an EC50 of 5 microM. 4. In the absence of GABA, propofol (10 microM-1 mM) activated small inward currents with a reversal potential similar to the Cl- equilibrium potential. The peak current amplitudes activated by propofol were only 31% of those activated by GABA in the same cells. 5. Like GABA (100 microM)-activated currents, propofol (100 microM)-activated currents were inhibited by the GABAA receptor antagonist, bicuculline (10 microM) and were abolished by Zn2+ (100 microM). 6. Propofol (10, 30 and 100 microM) dose-dependently activated currents in the absence of GABA. However, the peak amplitude of currents activated by propofol declined with concentrations > 100 microM. The cessation of application of a high dose of propofol (1 mM) was associated with a current 'surge'. 7. The surge current, seen after application of propofol (1 mM), had a reversal potential similar to the Cl- equilibrium potential. The ratio between peak current amplitude in the presence of propofol (1 mM) and surge current amplitude after propofol application, were not dependent on holding potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bidirectional effects of the neuroactive steroid tetrahydrodeoxycorticosterone on GABA-activated Cl- currents in cultured rat hypothalamic neurons.

1. The non-genomic effects of tetrahydrodeoxycorticosterone (THDOC; 5-alpha-pregnane-3-alpha, 21-diol-20-one) were studied in cultured hypothalamic neurons of the rat. 2. The effects of THDOC (10 nM - 1 microM) on responses to different concentrations of exogenously applied GABA and on spontaneous inhibitory postsynaptic currents (IPSCs) were measured with whole-cell voltage clamp recordings. 3. Application of GABA induced inward currents with dose-dependently increasing amplitudes (up to 3.9 nA at a holding potential of -20 mV). High doses of THDOC (100 nM-1 microM) induced small inward currents on its own (14+/-3 and 24+/-3 pA, respectively). 4. Simultaneous application of 10 microM GABA with 100 nM or 1 microM THDOC increased current amplitudes by 125 and 128%, respectively. At 10 nM THDOC exerted no consistent effects on GABA currents. 5. Responses to 1 microM of GABA were modulated in a bidirectional manner by different doses of THDOC: 10 nM THDOC reduced the amplitude of GABA responses to 80% (P=0.018, n=15), whereas 100 nM and 1 microM THDOC enhanced the GABA response to 115 and 180% (P=0.0007, n = 15), respectively. 6. The time constant of decay of spontaneous inhibitory postsynaptic currents (IPSCs) was reversibly increased from 91+/-10 to 314+/-34 ms (n=3) by the application of THDOC (1 microM). The amplitudes of the IPSCs were not affected by THDOC. 7. These data indicate that THDOC modulates GABA responses of hypothalamic neurons in a bidirectional manner, resulting in a complex tuning of neuronal excitability in the hypothalamus.     

Allosteric modulation of an expressed homo-oligomeric GABA-gated chloride channel of Drosophila melanogaster.

1. Functional GABA-gated chloride channels are formed when cRNA encoding the Drosophila melanogaster GABA receptor subunit RDL is injected into the cytoplasm of Xenopus oocytes. Two-electrode voltage-clamp was used to investigate allosteric modulation of GABA-induced currents recorded from the expressed, bicuculline-insensitive, RDL homo-oligomers. 2. Flunitrazepam (0.1 microM to 100 microM) had no effect on the amplitude of responses to 10 microM GABA (approximately EC10), whereas 4'chlorodiazepam (100 microM) enhanced the amplitude of submaximal responses to GABA. 3-Hydroxymethyl-beta-carboline (1 microM) and ethyl-beta-carboline-3-carboxylate (both 1 and 100 microM) had no effect on currents induced by 30 microM (approximately EC50) GABA. However 100 microM 3-hydroxymethyl-beta-carboline reduced potentiation by 4'chlorodiazepam. 3. The sodium salts of pentobarbitone (10 microM to 1 mM) and phenobarbitone (50 microM to 1 mM) dose-dependently enhanced submaximal GABA responses. Neither barbiturate activated currents in the absence of GABA. 4. At 10 microM, the steroids 5 alpha-pregnan-3 alpha-ol-20-one and alphaxalone (5 alpha-pregnan-3 alpha-ol-11,20-dione), potentiated submaximal GABA responses. The stereoselectivity of steroid action seen on vertebrate GABAA receptors was observed on RDL homo-oligomers as 5 alpha-pregnan-3 beta-ol-20-one (10 microM) was without effect. None of the three steroids tested activated currents in the absence of GABA. 5. The novel anticonvulsant, loreclezole (100 microM), potentiated the response to 10 microM GABA, but not that of saturating concentrations of GABA. delta-Hexachlorocyclohexane (0.1 microM to 30 microM) was a potent enhancer of submaximal responses to GABA of RDL. 6. The potencies of barbiturates and steroids on RDL homo-oligomers resemble those observed for several in situ insect GABA receptors, whereas those of benzodiazepine binding-site ligands are considerably reduced. The differences in the benzodiazepine pharmacology of RDL homo-oligomers and native GABA receptors, may reflect roles of other subunits in native insect receptors.     

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hexachlorocyclohexanes on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina.

Poly(A)+ RNA from rat cerebral cortex expresses gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, mediated by GABAA receptors (IG-Actx). In contrast, RNA from bovine retina expresses GABA responses composed of two pharmacologically distinct Cl- currents, one mediated by GABAA receptors (IG-Aret) and the other by atypical GABA receptors that are resistant to bicuculline and are not activated by baclofen (IG-BR). The pharmacology of the bicuculline/baclofen-insensitive GABA receptors was further investigated by comparing actions of hexachlorocyclohexane (HCH) enantiomers on GABA-activated membrane currents expressed in oocytes by brain and retina RNA. gamma-HCH (lindane) was a potent inhibitor of IG-Actx, with suppression of currents detectable at concentrations as low as 50 nM. The IC50 for gamma-HCH, calculated from inhibitory effects on maximum IG-Actx (current elicited by 3 mM GABA), was 7.3 +/- 3 microM. Inhibitory effects of gamma-HCH on IG-Aret were qualitatively similar to those described for IG-Actx. In contrast, alpha-HCH and delta-HCH induced clear positive modulation of IG-Actx elicited by low (e.g., 10 microM) concentrations of GABA. Thresholds for the modulatory effects of alpha-HCH and delta-HCH were between 100 and 300 nM, with maximum levels of potentiation (5-7-fold) between 20-50 microM. Potentiation of IG-Actx by alpha- and delta-HCH was reversible and largely insensitive to the benzodiazepine antagonist flumazenil (1 microM). Assays on maximum IG-Actx indicated that alpha-HCH (10-100 microM) caused only marginal reductions in response (less than or equal to 15%), whereas delta-HCH had stronger inhibitory effects (IC50, 20-30 microM). At concentrations between 0.1 and 50 microM, beta-HCH induced only 10-25% facilitation of IG-Actx elicited by 10 microM GABA and had no clear effects on maximum responses. IG-BR was also potently inhibited by gamma-HCH. Thresholds for detecting reductions in current were approximately 20 nM, and the IC50 calculated from effects on maximum responses was 5.8 +/- 2 microM. However, neither alpha-HCH nor delta-HCH (1-100 microM) induced any potentiation of IG-BR. alpha-HCH had some weak inhibitory effects that were largely surmountable, whereas delta-HCH and beta-HCH were essentially inactive. These experiments raise the possibility that alpha- and delta-HCH constitute a novel class of GABAA receptor modulators, which might prove to be useful for investigating the mechanisms underlying regulation of GABAA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mechanism of steroid anaesthetic (alphaxalone) block of acetylcholine-induced ionic currents.

The effects of the steroid anaesthetic alphaxalone on acetylcholine (ACh)-induced ionic channels were studied in voltage clamped 'myoballs' in culture. Alphaxalone produced a reversible blockade of the ACh-evoked inward current, ED50 = 6.0 microM. The ACh reversal potential (-5.0 mV), the single channel conductance (13.5 pS) and mean open time (3.6 ms) were unchanged by the drug. Thus, alphaxalone produced an 'all or none' block of the ionic channel. In double pulse conditioning experiments, alphaxalone produced an additional inhibition with a time constant of recovery (550 ms) much longer than the time constant of recovery of the normal desensitization (250 ms). It was concluded that alphaxalone blocks active (open) ionic channels.     

Effects of antagonists on quisqualate and nicotinic receptor-mediated currents of midbrain neurones in culture.

1. The action of non-N-methyl-D-aspartate (non-NMDA) and nicotinic antagonists on excitatory postsynaptic currents (e.p.s.cs) and on quisqualate (Quis)- and nicotine-gated currents was studied by use of whole-cell recording in dissociated culture of the rat midbrain. 2. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.1 microM) and kynurenic acid (0.1 mM) attenuated network-generated and miniature e.p.s.cs while mecamylamine (100 microM) and hexamethonium (400 microM) had no effect. Acetylcholine (ACh) enhanced or suppressed e.p.s.cs. The suppressing effect of ACh was blocked by atropine (0.1-10 microM). 3. ACh (50-1000 microM) and quisqualate (Quis, 0.1-20 microM) induced inward currents with the same reversal potential as e.p.s.cs. 4. Application of Quis and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) in a low concentration (0.5 and 5 microM, respectively) evoked a maintained current which was attenuated by CNQX (1 microM) and kynurenic acid (0.5 mM) but not by mecamylamine (100 microM). 5. Higher concentrations of Quis (5-20 microM) and AMPA (50-100 microM) evoked a transient and a maintained current component. Kynurenic acid (1 mM) reduced the transient but not the maintained component. CNQX (5-10 microM) increased the maintained component without reducing the transient one; 20 microM CNQX reduced both components. 6. ACh-induced transient current was mimicked by nicotine and reversibly and dose-dependently blocked by mecamylamine. Atropine (10 microM), hexamethonium (400 microM) as well as CNQX (100 microM) and kynurenic acid (1 mM) did not affect the current.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of action and pharmacological specificity of the anticonvulsant NMDA antagonist MK-801: a voltage clamp study on neuronal cells in culture.

1. Some possible molecular mechanisms of action of the anxiolytic, anticonvulsant and neuroprotective agent MK-801 have been examined in 'whole-cell' voltage clamp recordings performed on rat hippocampal and cortical neurones, bovine adrenomedullary chromaffin cells and N1E-115 neuroblastoma cells maintained in cell culture. 2. Transmembrane currents recorded from rat hippocampal and cortical neurones in response to locally applied N-methyl-D-aspartate (NMDA) were antagonized by MK-801 (0.1-3.0 microM). Blockade was use-dependent, and little influenced by transmembrane potential. MK-801 (3 microM) had no effect on currents evoked by kainate (100 microM). 3. The antagonism of NMDA-induced currents by MK-801 was only slowly and incompletely reversed when the cell membrane potential was clamped at -60 mV during washout. Prolonged applications of NMDA at +40, but not -60 mV during washout, markedly accelerated recovery from block. 4. In contrast to MK-801, ketamine (10 microM) blocked NMDA-induced currents in a voltage-dependent manner. Blockade increased with membrane hyperpolarization and was completely reversible upon washout. 5. MK-801 (1-10 microM) produced a voltage- and concentration-dependent block of membrane currents elicited by ionophoretically applied acetylcholine (ACh) recorded from bovine chromaffin cells. The block was readily reversible upon washout. 6. gamma-Aminobutyric acidA (GABAA) receptor-mediated chloride currents of chromaffin cells were unaffected by MK-801 (1-100 microM). In contrast, such currents were potentiated by diazepam (1 microM). MK-801 (100 microM) had no effect on currents evoked by GABA on hippocampal neurones. 7. MK-801 (10 microM) had little effect on membrane currents recorded from N1E-115 neuroblastoma cells in response to ionophoretically applied 5-hydroxytryptamine (5-HT). Such currents were antagonized by the 5-HT3 receptor antagonist GR 38032F (1 nM) and also by MK-801 at high concentration (100 microM). 8. Voltage-activated, tetrodotoxin-sensitive, sodium currents of chromaffin cells were unaffected by 10 microM MK-801. However, at a relatively high concentration (100 microM), MK-801 reduced the amplitude of such currents to approximately 77% of control. 9. The relevance of the present results to the central actions of MK-801 is discussed.     

Agonist and potentiation actions of n-octanol on gamma-aminobutyric acid type A receptors.

The n-octanol effects on the gamma-aminobutyric acid type A (GABAA) receptor were studied in human embryonic kidney 293 cells transfected with alpha1, beta2, and gamma2S subunit cDNAs. GABA-evoked currents had an EC50 of 13.3 +/- 1.7 microM and a Hill coefficient (nH) of 1.4 +/- 0.1. n-Octanol was also capable of evoking a small current with an EC50 of 1000 microM and an nH of 2. In addition, n-octanol modulated GABA-induced currents in a concentration-dependent manner. Coapplications of n-octanol increased peak currents evoked by 3 microM GABA with an EC50 of 190 microM and an nH of 1.8. The extent of potentiation decreased with increasing GABA concentrations and no potentiation was observed when n-octanol was coapplied with 1000 microM GABA. One-minute preapplication of 1000 microM n-octanol slightly potentiated 3 microM GABA-induced current, whereas it suppressed 300 microM GABA-induced current to 16% of the control, suggesting that 84% of the receptors underwent desensitization. Two models were used to explain n-octanol agonistic and potentiating actions on the alpha1beta2gamma2S GABAA receptor: n-octanol binds to multiple sites to exert multiple actions, or n-octanol acts as a partial agonist to manifest these actions. The partial agonist model is unique because it is a simpler model to explain n-octanol actions on the GABAA receptor.     

Substance P modulates the sensitivity of the nicotinic receptor in amphibian cholinergic transmission.

The effect of substance P on the sensitivity of nicotinic acetylcholine (ACh) receptors of bullfrog sympathetic ganglion cells and frog skeletal muscle endplate was examined electrophysiologically. The amplitude of ACh-induced postsynaptic potential (ACh potential) and current (ACh current) were reversibly and dose-dependently reduced by substance P at low concentrations (0.42-42 microM). The mean amplitude of the miniature endplate potential (m.e.p.p.) was also reduced by substance P (4.2 microM). Substance P (4.2 microM) shifted the S-shaped dose-response curve of the ACh current downward. A Lineweaver-Burk plot constructed from the dose-response curve revealed that substance P depressed the maximum response (Vmax) without changing the apparent affinity (Km) of ACh for the receptor. Substance P (0.42-42 microM) did not alter the reversal potential of the ACh current of the endplate. The half-decay time of endplate current (e.p.c.) and its voltage-dependency were not altered by substance P in these concentrations. The depression of the ACh current by substance P may not be due to a blockade of the opened channel which has been activated by the preceding combination of ACh with the receptor. These results suggest that substance P suppresses the sensitivity of nicotinic ACh-receptors of the sympathetic ganglion cell and skeletal muscle endplate, acting on a certain allosteric site but not the recognition site of ACh in the receptor-ionic channel complex.     

Modulation of GABAA receptor-mediated currents by phenazepam and its metabolites

The effects of 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam, PNZ), a 1,4-benzodiazepine derivative, and its physiological metabolites on GABA-activated whole-cell currents were studied in enzymatically isolated rat Purkinje neurones. PNZ, its hydroxylated metabolite (HPNZ) and a reference benzodiazepine, diazepam, potently enhanced (up to 200% of control) peak amplitude of currents activated by 10 microM GABA with EC50s of 6.1 +/- 0.8, 10.3 +/- 1.4 and 13.5 +/- 1.9 nM respectively. Both PNZ and HPNZ caused a parallel leftwards shift of the concentration/effect relationship for GABA. Another metabolite, 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ), augmented responses to 10 microM GABA with a maximal efficacy similar to that of the 1,4benzodiazepines tested, although its EC50 was 2.4 +/- 0.2 microM. A further metabolite, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone (ABPH), had only minimal effects on the responses elicited by 10 microM GABA. Incubation with QNZ and ABPH had biphasic effects on the concentration/effect relationship for GABA. These compounds enhanced peak amplitudes of currents activated by low concentrations of GABA, but inhibited responses to saturating concentrations of the agonist. This effect could, in part, be explained by the acceleration of the desensitisation process by those substances. It is concluded that both PNZ and HPNZ can be referred to as full positive modulators of GABAA receptors and that they are primarily responsible for GABAergic effects of therapeutic doses of PNZ.     

The tremorigen aflatrem is a positive allosteric modulator of the gamma-aminobutyric acidA receptor channel expressed in Xenopus oocytes

Aflatrem, a mycotoxin from Aspergillus flavus, potentiates the gamma-aminobutyric acid (GABA)-induced chloride current. This positive allosteric regulatory action of aflatrem was quantitatively studied on the GABAA receptor channel expressed in Xenopus oocytes after injection with chick brain mRNA under voltage-clamp conditions. In this model system, aflatrem potentiates the current induced by 5 microM GABA in a concentration-dependent manner. Half-maximal potentiation was obtained with 2.4 microM aflatrem and maximal stimulation of the GABA (5 microM) response was more than 10-fold. The potentiation was not associated with a change of the reversal potential of the GABA-induced current. In the presence of 2 microM aflatrem, the GABA dose-response curve shifted to lower concentrations, with the Ka decreasing from 28 to 7 microM and the Hill coefficient, n, from 1.5 to 0.8, as measured at a membrane potential of -100 mV. At saturating concentration of GABA (250 microM), aflatrem (10 microM) was still able to enhance the current by about 21%. Further experiments suggest that the site of action of aflatrem on the GABAA receptor channel complex is different from that of benzodiazepines, pentobarbital, and picrotoxin. Aflatrem (10 microM) had no significant effect on the coexpressed voltage-dependent sodium and calcium channels and on the kainate channel. The potentiating action of aflatrem on the GABAA receptor channel may explain the initial symptoms of intoxication caused by aflatrem in vivo, i.e., diminished activity or immobility of the affected animal.     

Galphimine B modulates synaptic transmission on dopaminergic ventral tegmental area neurons

Galphimine B (GB) is a bioactive compound isolated from the plant Galphimia glauca Cav. (Malpighiaceae) and has been shown to have central nervous system depressant properties. In an earlier study, it was reported that both systemic and local administration of GB modified the extracellular spontaneous spiking activity in the ventral tegmental area (VTA) neurons. In the present study we analyzed the synaptic effects of this compound on dopaminergic neurons. Recordings were made in brain slices using intracellular and patch-clamp techniques, in dopaminergic (DA) VTA neurons. Spontaneous miniature excitatory postsynaptic currents (mEPSCs), excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic potentials (IPSPs) were recorded in control situation and after bath infusions of several concentrations of GB (1 microM-5 mM), GABA (1 nM-100 microM) and the GABA A blockers, picrotoxin (100 microM) and bicuculline (10 microM), and GABA B blocker saclofen (200 microM). GB administration reduced the frequency (p < 0.05) but not the amplitude of mESPCs. However, GABA (IC 50 = 645 nM) and GB (IC 50 = 174.5 microM) infusion significantly reduced the amplitude of stimuli induced EPSCs, Bicuculline (10 microM) co-administration only reduced GABA effects and did not modify the GB depressant action. Finally, isolated GABAergic IPSPs were modified by the addition of picrotoxin, but GB had no effect on these evoked synaptic responses. The present results indicate that GB modifies synaptic activity on dopaminergic VTA neurons by a non-GABAergic mechanism.     

Alphaxalone,a steroid anesthetic,inhibits the startle-enhancing effects of corticotropin releasing factor,but not strychnine

Corticotropin releasing factor (CRF) is a 41 amino acid peptide implicated in the expression of stress-and fear-enhanced behaviors. CRF potentiates the amplitude of the startle reflex, and this effect is reversed by benzodiazepines (BDZ), suggesting that the startle-enhancing effects of CRF are modulated by changes in the GABA/BDZ receptor complex. In the present study, CRF-potentiated startle is inhibited by alphaxalone, a pregnane steroid anesthetic that is thought to act via the GABA/BDZ receptor complex. Alphaxalone (ALX) does not reduce CRF-potentiated startle by producing a generalized reduction in reactivity, since blockade of CRF-stimulated startle was not accompanied by an ALX-induced reduction in baseline startle amplitude, and ALX does not reduce strychnine-potentiated startle. The effects of alphaxalone on CRF-potentiated startle may not be generalized to all CRF-stimulated behaviors, since alphaxalone failed to disrupt CRF-stimulated locomotor activity. CRF-potentiated startle is a useful assay for studying the effects of novel anxiolytic agents, and alphaxalone appears to be a steroid anesthetic with anxiolytic properties in this assay.     

GABA preincubation of rat brain sections increases [3H]GABA binding to the GABAA receptor and compromises the modulatory interactions

Receptor autoradiography has been employed to investigate the effect of gamma-aminobutyric acid (GABA) preincubation on the interaction of the GABAA receptor with its ligands. [3H]GABA (50 nM) binding to the GABAA receptors is increased by 60% compared to control sections after GABA (100 microM) preincubation. Receptor autoradiography shows that the increase is more pronounced in certain brain areas. The allosteric interactions between the GABA and benzodiazepine recognition sites were also examined. An increase in [3H]GABA (50 nM) binding to rat brain sections by co-incubation with the benzodiazepine, flunitrazepam (FNZ) has been observed autoradiographically. This effect has been quantitated in several brain regions; the overall brain increase in [3H]GABA binding induced by 1 microM FNZ was 20%. The increase in [3H]FNZ (1 nM) binding by co-incubation with GABA has also been observed autoradiographically, and the effect quantitated in four brain regions. The overall brain increase in [3H]FNZ binding induced by 100 microM GABA was 34%. After GABA preincubation these allosteric responses are significantly reduced in size. The increase in the [3H]GABAA binding as a consequence of GABA preincubation appears to reflect an increase in receptor affinity for [3H]GABA with no significant change in the maximum number of binding sites. We suggest that GABA preincubation converts the GABAA receptor to a higher affinity desensitised receptor conformation.     

Trichloroethanol potentiation of gamma-aminobutyric acid-activated chloride current in mouse hippocampal neurones.

1. The action of 2,2,2-trichloroethanol on gamma-aminobutyric acid (GABA)-activated Cl- current was studied in mouse hippocampal neurones in tissue culture by use of whole-cell patch-clamp recording. 2. Trichloroethanol increased the amplitude of currents activated by 1 microM GABA or 0.1 microM muscimol. Trichloroethanol, 1-25 mM, potentiated current activated by 1 microM GABA in a concentration-dependent manner with an EC50 of 3.0 +/- 1.4 mM and a maximal response (Emax) of 576 +/- 72% of control. 3. Trichloroethanol potentiated currents activated by GABA concentrations < 10 microM, but did not increase the amplitude of currents activated by concentrations of GABA > or = 10 microM. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration-response curve. 4. Trichloroethanol, 5 mM, potentiated GABA-activated current in neurones in which ethanol, 10-500 mM, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15-4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5. In the absence of exogenous GABA, trichloroethanol at concentrations > or = 2.5 mM activated a current that appeared to be carried by Cl- as its reversal potential changed with changes in the Cl- gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6. Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA-activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.     

Pumiliotoxin-C and synthetic analogues. A new class of nicotinic antagonists

Pumiliotoxin-C (PTX-CI), a cis-decahydroquinoline alkaloid, and synthetic analogues inhibited directly and indirectly elicited twitches in rat phrenic nerve diaphragm preparations. At 40 microM the most potent analogue, 2,5-di-n-propyl-cis-decahydroquinoline (PTX-CII), reversibly blocked the indirectly elicited twitch and markedly depressed the amplitude of end-plate potentials and miniature end-plate potentials without affecting muscle membrane potential. PTX-CII did not depress the directly elicited twitch in diaphragm muscle treated with alpha-bungarotoxin. At 40 microM, the compound blocked extrajunctional acetylcholine (ACh) sensitivity in chronically denervated soleus muscle of the rat. The peak amplitude of the end-plate current in frog sartorius muscle was depressed by PTX-CII at both positive and negative membrane potentials. Nonlinearity in the current-voltage relationship appeared at membrane potentials greater than -100 mV with concentrations of PTX-CII from 2.5 to 7.5 microM. The decay phase of the end-plate current was shifted to lower values such that it decayed faster at all membrane potentials but retained voltage sensitivity. PTX-CII, the most potent of the analogues, inhibited binding of [3H]perhydrohistrionicotoxin to Torpedo electroplax membranes with an IC50 value of 1.5 microM in the absence of carbamylcholine (Carb) and 0.1 microM in the presence of Carb. The other analogues were less potent, but all showed higher affinities in the presence of Carb. PTX-CI, PTX-CII, and PTX-CIII had no effect on the binding of [125I]alpha-bungarotoxin to ACh receptors in Torpedo membranes while slightly enhancing the binding of [3H]ACh, whereas PTX-CIV gave slight inhibition of both receptor ligands at concentrations greater than 10 microM. The pumiliotoxin-C class of alkaloids therefore appears to block neuromuscular transmission primarily via interactions with sites associated with the ion channel controlled by the ACh receptor.     

Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)