Comparison of the toxicokinetics of the convulsants picrotoxinin and tetramethylenedisulfotetramine (TETS) in mice.

Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 µM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2ß3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.

Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ-Aminobutyric Acid Type A Receptor (GABAAR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability.

Recent reports indicate that α6ß2/3γ2 GABAAR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6ß2/3γ2 GABAAR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABAAR α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6ß2/3γ2 GABAARs and were functionally silent at diazepam sensitive α1ß2/3γ2 GABAARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABAAR α6ß2/3γ2 subtypes.

Imaging MALDI MS of Dosed Brain Tissues Utilizing an Alternative Analyte Pre-extraction Approach.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry has been adopted in the pharmaceutical industry as a useful tool to detect xenobiotic distribution within tissues. A unique sample preparation approach for MALDI imaging has been described here for the extraction and detection of cobimetinib and clozapine, which were previously undetectable in mouse and rat brain using a single matrix application step. Employing a combination of a buffer wash and a cyclohexane pre-extraction step prior to standard matrix application, the xenobiotics were successfully extracted and detected with an 8 to 20-fold gain in sensitivity. This alternative approach for sample preparation could serve as an advantageous option when encountering difficult to detect analytes.

Characterization of the picrotoxin site of GABAA receptors.

This unit describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics, and competition assays (IC50, Ki determination) are included.

Evidence that clozapine directly interacts on the GABAB receptor.

Neurophysiological studies suggest that clozapine may facilitate γ-aminobutyric acid (GABAergic) neurotransmission. Therefore, we studied the interaction between clozapine and the GABAB receptor (GABABR). We showed that clozapine, and not N-desmethylclozapine, which is a metabolite of clozapine, increased the binding of the GABABR antagonist, [³H]-CGP54626A, at GABABRs. Linear regression analysis showed that the correlation between the dose of clozapine and the increase of [³H]-CGP54626A binding was significant. The curve of specific [³H]-CGP54626A binding in competition with different concentrations of GABA was left shifted in the presence of clozapine. With HEK293 cells overexpressing GABABR, we showed that clozapine had a significant increase of [³H]-CGP54626A binding at GABABR1 subunit, which provided a clue of the potential therapeutic target of clozapine.

GABAergic dysfunction in mGlu7 receptor-deficient mice as reflected by decreased levels of glutamic acid decarboxylase 65 and 67kDa and increased reelin proteins in the hippocampus.

Glutamate is the main excitatory neurotransmitter in the brain, while gamma-aminobutyric acid (GABA) is a primary inhibitory neuromodulator. Both amino acids act through ionotropic and metabotropic receptors that are widely distributed in the central nervous system. There are at least eight subtypes of metabotropic glutamate receptors (mGlu), which have been divided into three groups (mGlu I, II, and III). The mGlu7 receptor subtype, which belongs to the mGlu III group, seems to play a special role, as it is abundant in brain structures that are known to be responsible for antidepressant and/or anxiolytic activity of drugs. In GABAergic neurons, GABA is synthesised from glutamate by the pyridoxal phosphate (PLP)-dependent enzyme glutamic acid decarboxylase (GAD). It is expressed as two major isoforms, GAD65 and GAD67, responsible for the synthesis of the vesicular and cytoplasmic pool of neurotransmitter, respectively. Moreover, GABAergic neurons express a variety of proteins such as reelin, involved in synaptic transmission and plasticity. The aim of our study was to investigate the regulation of GABA synthesis and the level of modulatory receptor for GABA in mice lacking mGlu7 receptor for glutamate. The levels of GAD mRNA, GADs, and reelin proteins in the hippocampi of mGlu7-/- and mGlu7-/+ mice were measured using in situ hybridisation, immunohistochemistry, and Western blotting (WB). GAD mRNAs in the CA and DG regions of the hippocampus were measured separately. The levels of GAD65, GAD67, and reelin proteins were determined in the homogenates using WB, and the number of stained neurons was estimated using a stereological method of counting. GABA(B) receptor level was measured using a radioligand binding assay. Our results show that the mRNA and protein levels of both GADs were decreased in the hippocampi of animals lacking the mGlu7 receptor. Decreased levels of GAD67 mRNA were found in both the CA and DG regions, while the decrease in GAD65 mRNA was observed mainly in the CA region of the hippocampus. The protein levels of GAD65 was lowered in mGlu7-/- animals only, while GAD67 and GABA(B) receptor number were decreased in both mGlu7+/- and mGlu7-/- mice when measured in the whole hippocampus. In contrast, reelin was shown to be increased both in mGlu7-/+ and mGlu7-/- mice. The results suggest that mGlu7 receptor is involved in the regulation of GABAergic system activity at the level of GABA synthesised enzymes, specific proteins expressed by GABAergic neurons and metabotropic receptor for GABA.

Behavioral deficit and decreased GABA receptor functional regulation in the cerebellum of epileptic rats: effect of Bacopa monnieri and bacoside A.

In the present study, the effects of Bacopa monnieri and its active component, bacoside A, on motor deficit and alterations of GABA receptor functional regulation in the cerebellum of epileptic rats were investigated. Scatchard analysis of [(3)H]GABA and [(3)H]bicuculline in the cerebellum of epileptic rats revealed a significant decrease in B(max) compared with control. Real-time polymerase chain reaction amplification of GABA(A) receptor subunits-GABA(Aalpha1), GABA(Aalpha5,) and GABA(Adelta)-was downregulated (P<0.001) in the cerebellum of epileptic rats compared with control rats. Epileptic rats exhibit deficits in radial arm and Y-maze performance. Treatment with B. monnieri and bacoside A reversed these changes to near-control levels. Our results suggest that changes in GABAergic activity, motor learning, and memory deficit are induced by the occurrence of repetitive seizures. Treatment with B. monnieri and bacoside A prevents the occurrence of seizures thereby reducing the impairment of GABAergic activity, motor learning, and memory deficit.

Dissociation constants for GABA(A) receptor antagonists determined with neuronal networks on microelectrode arrays.

Changes in spontaneous spike activities from murine frontal cortex networks grown on microelectrode arrays were used to determine the dissociation constants of three GABA(A) antagonists: gabazine, bicuculline, and trimethylolpropane phosphate (TMPP). Networks were treated with fixed concentrations of antagonists and titrated with the GABA(A) receptor agonist muscimol. Muscimol decreased spike activity in a concentration-dependent manner with full efficacy (100% spike inhibition). A sigmoidal curve fit provided a 50% inhibitory concentration (IC(50)) of 0.14+/-0.05muM (mean+/-S.D., n=5). Increasing concentrations of the three antagonists shifted the muscimol concentration response curves (CRCs) to the right with the same efficacy. Schild plot analyses with linear regressions resulted in slopes that are statistically not different from unity and provided X-intercepts (dissociation constants) of 0.23, 0.61, and 3.98muM for gabazine, bicuculline, and TMPP, respectively. Corresponding pA2 values (-logK(B)) were 6.64, 6.21, and 5.40. The dissociation constants for gabazine and bicuculline agree well with those obtained with other methods. The TMPP K(B) has not yet been reported in the literature. The data suggest that spontaneously active networks on microelectrode arrays can be used as reliable platforms for rapid quantitative pharmacological investigations.

Rat pharmacokinetics and pharmacodynamics of a sustained release formulation of the GABAA alpha5-selective compound L-655,708.

The pharmacokinetic and pharmacodynamic (i.e., receptor occupancy) properties of L-655,708, a compound with selectivity for alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors, were examined in rats with the aim of developing a formulation that would give sustained (up to 6 h) and selective occupancy of alpha5-containing GABA(A) receptors suitable for behavioral studies. Standard rat pharmacokinetic analyses showed that L-655,708 has a relatively short half-life with kinetics in the brain mirroring those in the plasma. In vivo binding experiments showed that plasma concentrations of around 100 ng/ml gave relatively selective in vivo occupancy of rat brain alpha5-versus alpha1-, alpha2-, and alpha3-containing GABA(A) receptors. Therefore, this plasma concentration was chosen as a target to achieve relatively selective occupancy of alpha5-containing receptors using s.c. implantations of L-655,708 (0.4, 1.5, or 2.0 mg) formulated into tablets of various size (20 or 60 mg) containing different amounts of L-655,708 and combinations of low and high viscosity hydroxypropyl methylcellulose (LV- and HV-HPMC). The optimum formulation, 1.5 mg of L-655,708 compressed into a 60-mg tablet with 100% HV-HPMC, resulted in relatively constant plasma concentrations being maintained for at least 6 h with very little difference between C(max) concentrations (125-150 ng/ml) and plateau concentrations (100-125 ng/ml). In vivo binding experiments confirmed the selective occupancy of rat brain alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors.

A postnatal switch in GABAergic control of spinal cutaneous reflexes.

GABAergic signalling exerts powerful inhibitory control over spinal tactile and nociceptive processing, but during development GABA can be depolarizing and the functional consequences of this upon neonatal pain processing is unknown. Here we show a postnatal switch in tonic GABA(A) receptor (GABA(A)R) modulation of cutaneous tactile and nociceptive reflexes from excitation to inhibition, but only in the intact spinal cord. Neonatal and 21-day-old (P21) rats were intrathecally treated with one of the GABA(A)R antagonists bicuculline and gabazine, with both compounds dose-dependently decreasing hindpaw mechanical and thermal withdrawal thresholds in P21 rats but increasing them in P3 neonates. Intrathecal gabazine also produced an increase in the cutaneous evoked electromyography (EMG) response of the biceps femoris in P21 rates but lowering the response in neonates. Injections of 3H-gabazine in the L4-L5 region at P3 confirmed that gabazine binding was restricted to the lumbar spinal cord. Spinalization of P3 neonates at the upper thoracic level prior to drug application reversed the behavioural and EMG responses to GABA antagonists so that they resembled those of P21 rats. The effects of spinalization were consistent with gabazine facilitation of ventral root potentials observed in isolated neonatal spinal cord. These data show a marked postnatal developmental switch in GABAergic control of neonatal nociception that is mediated by supraspinal structures and illustrate the importance of studying developmental circuits in the intact nervous system.

2-[4-(3,4-Dimethylphenyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393), a selective dopamine D4 receptor antagonist.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

SGS-742 Novartis.

SGS-742, a GABA(B) antagonist, is being developed by Saegis, under license from Novartis, for the potential treatment of mild cognitive impairment and Alzheimer's disease (AD). In May 2004, Saegis began enrollment in a phase II trial of SGS-742 in mild-to-moderate AD patients.

Reproductive health effects and teratogenicity of antiepileptic drugs.

Women with epilepsy are less likely to bear children than women in the general population, and although this reduced fertility can be attributed in part to effects of the disease itself, the effects of antiepileptic drugs (AEDs), including changes in reproductive endocrine function, are also a factor. Conversely, some AEDs interact with oral contraceptives and can increase the risk for contraceptive failure and unplanned pregnancy. Women with epilepsy also have elevated rates of congenital anomalies and major malformations in their offspring, for which exposure of the developing fetus to AEDs taken by the mother appears to be responsible. In utero exposure to some AEDs may also be associated with increased risk for impaired cognitive function in the growing child. Clearly, possible long-term effects on reproductive health and pregnancy outcomes require careful attention when AED therapy is being considered for a patient with childbearing potential. Moreover, because AEDs are increasingly being used in therapy for other conditions such as migraine, bipolar disorder, and pain, it is not only the treatment of women with epilepsy that will be affected by these concerns.

4-Hydroxy-trans-2-nonenoic acid is a gamma-hydroxybutyrate receptor ligand in the cerebral cortex and hippocampus.

Elevated production of 4-hydroxy-trans-2-nonenal (HNE) occurs in numerous neurological disorders involving oxidative damage. HNE is metabolized to the non-toxic 4-hydroxy-trans-2-nonenoic acid (HNEAcid) by aldehyde dehydrogenases in the rat cerebral cortex. Based upon the structural similarity of HNEAcid to ligands of the gamma-hydroxybutyrate (GHB) receptor, we hypothesized that HNEAcid is an endogenous ligand for the GHB receptor. HNEAcid displaced the specific binding of the GHB receptor ligand (3)H-NCS382 (30 nm) in membrane preparations of human frontal cerebral cortex and whole rat cerebral cortex with IC(50s) of 3.9 +/- 1.1 and 5.6 +/- 1.2 micro m, respectively. Inhibition was attenuated when the carboxyl group of HNEAcid was replaced with an aldehyde or an alcohol. HNEAcid (300 micro m) did not displace the binding of beta-adrenergic receptor and GABA(B) receptor antagonists, demonstrating the selectivity of HNEAcid for the GHB receptor. HNEAcid is formed in homogenates of human frontal cortical gray matter in an NAD(+)-dependent (V(Max), 0.71 nmol/min/mg) and NADP(+)-dependent (V(Max), 0.12 nmol/min/mg) manner. Lastly, (3)H-NCS382 binding is elevated 2.7-fold with age in the cerebral cortex of rats. Our data demonstrate that an HNE metabolite, formed in rat and human brain, is a signaling molecule analogous to other bioactive lipid peroxidation products.

Changes in rectal temperature and ECoG spectral power of sensorimotor cortex elicited in conscious rabbits by i.c.v. injection of GABA, GABA(A) and GABA(B) agonists and antagonists.

1. In order to ascertain whether both GABA(A) and GABA(B), or only GABA(B) receptors, directly modulate thermoregulation in conscious rabbits, GABA(A)/GABA(B) agonist and antagonist agents were injected intracerebroventricularly in conscious rabbits while monitoring changes in rectal temperature (RT), gross motor behaviour (GMB) and electrocorticogram (ECoG) power spectra (ps) from sensorimotor cortices. 2. GABA (48 micromol), nipecotic acid (50 nmol), THIP (60 nmol), muscimol (18 nmol) and baclofen (8 nmol) induced hypothermia (-deltaRTmax values of 1.70+/-0.1, 1.4+/-0.2, 1.0+/-0.4, 1.1+/-0.2 and 1.6+/-0.3 degrees C, respectively), accompanied by inhibition of GMB and ECoG synchronization. THIP increased ps at delta frequency band (1.1-3.3 Hz), while GABA, nipecotic acid, muscimol and baclofen did the same at both delta and (4.6-6.5 Hz) frequency bands. ECoG ps changes were concomitant or even preceded hypothermia. 3. Bicuculline (1.8 nmol) induced hyperthermia (deltaRTmax 1.2+/-0.5 degrees C) and slight excitation of GMB, while CGP35348 (1.2 micromol) did not affect RT nor GMB. Both compounds did not affect ECoG ps. 4. Bicuculline potentiated muscimol-induced hypothermia, inhibition of GMB and synchronization of ECoG, while CGP35348 fully antagonized these effects. 5. In conclusion, the present results, while confirming the prevailing role of GABA(B), also outline a direct involvement of GABA(A) receptors in the central mechanisms of thermoregulation. Ascending inhibition towards discrete cortical areas controlling muscular activity and thermogenesis may result from GABA receptor activation in neurones proximal to the ventricles, thus contributing to hypothermia, although hypothermia-induced reduction of neuronal activity of these cortical areas cannot be ruled out.

On high- and low-affinity agonist sites in GABAA receptors.

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.

Proconvulsant-induced seizures in alpha(4) nicotinic acetylcholine receptor subunit knockout mice.

The genetic basis of a number of epilepsy syndromes has been identified but the precise mechanism whereby these mutations produce seizures is unknown. Three mutations of the alpha(4) subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified in autosomal dominant nocturnal frontal lobe epilepsy. In vitro studies of two mutations suggest an alteration of receptor function resulting in decreased ion channel current flow. We investigated the response of alpha(4) nAChR subunit knockout mice to the gamma-aminobutyric acid (GABA) receptor antagonists; pentylenetetrazole (PTZ) and bicuculline (BIC), the glutamate receptor agonist kainic acid (KA), the glycine receptor antagonist strychnine and the K(+) channel blocker 4-aminopyridine (4-AP). Mutant (Mt) mice had a greater sensitivity to PTZ and BIC, with an increase in major motor seizures and seizure-related deaths. Furthermore, Mt mice were more sensitive to KA and strychnine, but the effects were much smaller compared to those seen with the GABA receptor antagonists. Paradoxically, Mt mice appeared to be relatively protected from 4-AP-induced major motor seizures and death. The results show that a functional deletion of the alpha(4) nAChR subunit in vivo is associated with a major increase in sensitivity to GABA receptor blockers.

A single subunit (GB2) is required for G-protein activation by the heterodimeric GABA(B) receptor.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152-2159). In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca(2+) channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA(B) receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and G-protein activation, these two functions are assumed by two distinct subunits in the GABA(B) heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.

Brain microdialysis and PK/PD correlation of pregabalin in rats.

Pregabalin [PGB, (S)-3-isobutyl GABA, CI-1008] is a derivative of the inhibitory neurotransmitter g-aminobutyric acid (GABA). It has shown anticonvulsant, analgesia and anxiety activity in animal models. In this report, blood-brain barrier (BBB) influx and efflux of PGB were investigated with microdialysis at efficacious doses in rats. BBB influx (CLin) and efflux (CLout) permeability for pregabalin were 4.8 and 37.2 microL/min/g brain, respectively, following an intravenous infusion to rats. The results indicate that PGB is brain penetrable, supporting its anti-epilepsy and other CNS pharmacology. Significant anticonvulsant action of PGB was detected between 2 and 8 hr post oral dose, which is lag behind ECF drug concentrations lees. A PK/PD link model was used to describe the counter-clockwise hysteresis relationship between pregabalin brain ECF concentration and the anticonvulsant effect in rats. The resulting Ce (concentration in effect compartment) versus effect profile exhibits a sigmoidal curve and the calculated ECe50 and Keo values were 95.3 ng/mL and 0.0092 min-1, respectively. The small Keo value suggests that the effect is not directly proportional to the amount of pregabalin in the ECF compartment possibly due to inherent delay.

Neither GABA(A) nor strychnine-sensitive glycine receptors are the sole mediators of MAC for isoflurane.

UNLABELLED: Inhaled anesthetics produce immobility (a cardinal aspect of general anesthesia) by an action on the spinal cord, possibly by potentiating the responses of gamma-amino-n-butyric acid (GABA(A)) and glycine receptors to GABA and glycine. In this study, we antagonized GABA(A) and glycine responses by intrathecal administration of picrotoxin (a noncompetitive GABA(A) antagonist), strychnine (a competitive glycine antagonist), or combinations of these drugs. We measured the capacity of antagonist infusion to increase isoflurane MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). We found that these potent GABA(A) and glycine receptor antagonists had a ceiling effect, either alone or in combination increasing the MAC of isoflurane by at most 47%. IMPLICATIONS: gamma-amino-n-butyric acid and glycine receptors may in part be responsible for the immobilizing action of isoflurane. They are not, however, the only receptors that contribute to isoflurane-induced immobility (i.e., that determine the MAC of isoflurane).