Solubilization of Active Brain α1-Adrenoceptors by a Zwitterionic Detergent

Solubilization of rat brain alpha 1-adrenoceptors was performed by treatment with 6 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio] - 1 - propanesulfonate). The alpha 1-adrenoceptor antagonist [3H]prazosin was shown to bind reversibly and specifically to the soluble extract obtained after centrifugation at 150,000 X g for 1 h. Separation of the soluble [3H]prazosin-bound complexes was performed by the polyethylene glycol precipitation technique followed by filtration. A Scatchard plot of the concentration-dependent binding curve showed only one class of binding sites, with a high affinity for [3H]prazosin. Affinity of the solubilized receptors for the ligand increased as the CHAPS concentration in the assay medium decreased; the number of binding sites remained unchanged (approximately equal to 70 fmol/mg protein). This corresponds to a 30% recovery of original membrane sites. The solubilized receptors presented the same characteristics of specificity and stereospecificity as membrane alpha 1-adrenoceptors. Moreover, 150 mM NaCl was found to modulate the affinity of epinephrine for the [3H]prazosin-bound soluble complex, as previously described for membrane preparations. Thus, CHAPS appears to be a suitable detergent for solubilizing rat brain alpha 1-adrenoceptors and preserving their functional activities.     

Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000-650,000 by gel filtration, the sedimentation coefficients being 11.0-11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for flunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in th hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.     

Solubilization of Adrenal Angiotensin II Receptors with a Zwitterionic Detergent

Abstract

Active rat adrenal angiotensin II receptors have been solubilized with a zwitterionic detergent, 3 [(3-cholamidopropyl)-dimethylammonio] -1-propane sulfonate. The solubilized receptors retain a high affinity for angiotensin II (Kd = 1.9 nM) similar to the value observed in adrenal membrane particles (Kd = 1.2 nM). In addition, the binding-inhibition potency of several angiotensin II peptides is maintained unaltered, indicating a fully preserved specificity of the solubilized receptors.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Ethylenediamine and GABA Potentiation of [3H]Diazepam Binding to Benzodiazepine Receptors in Rat Cerebral Cortex

Specific binding of [3H]diazepam at a free concentration of 2 nM was found to be maximally potentiated by 117% in Tris-HCl buffer and 160% in Tris-citrate buffer by ethylenediamine (EDA), but only at relatively high concentrations of EDA (ED50 = 5 X 10(-5) M), although this potentiation was susceptible to a low dose (6 microM) of bicuculline. Dose-response curves show that EDA differs from GABA with respect to both potency and efficacy. In additivity experiments no evidence was found that EDA could act as a partial agonist at GABA receptors, and it was concluded that EDA and GABA apparently do not potentiate [3H]diazepam binding by acting on the same receptor. Scatchard analysis lends support to this hypothesis, indicating that the potentiation of [3H]diazepam binding by 3.16 X 10(-3) M EDA is due to an increase in receptor number (from 930 to 1170 fmol/mg protein) and not receptor affinity (remaining constant about 20 nM). Subsequent studies showed the potentiation to be reversible. It is concluded that EDA can act on the GABA-benzodiazepine receptor ionophore complex but that this is probably not a direct action on the GABA receptor. It is suggested that EDA can be used to differentiate GABA receptors linked to benzodiazepine receptors from those not so linked.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t-Butylbicyclophosphorothionate Binding Site Within the γ-Aminobutyric Acid Receptor-Ionophore Complex

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.     

Heterogeneity of Benzodiazepine Receptors in the Central Nervous System Demonstrated with Kenazepine, an Alkylating Benzodiazepine

Binding studies using the alkylating benzodiazepine kenazepine strongly suggest the existence of several populations of benzodiazepine receptors in the CNS. Kenazepine reacts noncompetitively and irreversibly with some receptors and competitively (reversibly) with others. Cerebellum contains the largest proportion (approx. 80%) of the noncompetitive type, while hippocampus and cortex contain a preponderance of competitive-type receptors (approx. 80 and 50%, respectively). The Hill coefficients for kenazepine are approx. 0.7 in cortex and cerebellum, and near unity in dorsal hippocampus. Different populations of benzodiazepine receptors may mediate different physiologic and pharmacologic effects in vivo.     

Molecular and Pharmacological Characterization of GABAA Receptors in the Rat Pituitary

Abstract: Levels of mRNA for the major subunits of the GABA_A receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [³H]muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 n M , respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [³H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 n M in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABA_A receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

In Vitro Modulation by Avermectin B1a of the GABA/Benzodiazepine Receptor Complex of Rat Cerebellum

Avermectin B1a, a macrocyclic lactone anthelmintic agent, causes a concentration-dependent increase of [3H]flunitrazepam binding to membranes from rat cerebellum by increasing the affinity and the number of binding sites. This effect appears to be independent of the concentration of chloride ions. The effects of avermectin B1a occur with high affinity (EC50 = 70 nM), and they persist after washing of the membranes with drug-free buffer. Pretreatment of the membranes with Triton X-100 completely abolishes the action of avermectin B1a. GABA and the GABA-mimetic compounds piperidine-4-sulfonic acid and THIP diminish the effects of avermectin B1a on benzodiazepine receptor binding in a bicuculline-methiodide-sensitive mode. In addition, the stimulation of [3H]flunitrazepam binding by avermectin B1a is decreased by the pyrazolopyridines etazolate and cartazolate. These observations suggest that avermectin B1a stimulates benzodiazepine receptor binding by acting on a modulatory site which is independent of the GABA recognition site and of the drug receptor for the pyrazolopyridines, but which is in functional interaction with these sites.     

Enhancement of γ-Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors

We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.     

Characterization of the Influence of Unsaturated Free Fatty Acids on Brain GABA/Benzodiazepine Receptor Binding In Vitro

Abstract: 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°C) of the binding assay and not additive to the enhancement by high concentrations of C1. In rat brain preparations, the stimulation of [³H]diazepam binding by oleic acid (10^?4M) 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^?4M) 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^?4M) 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 ～200,000 daltons. Our data show that unsaturated FFAs have distinct effects on membranebound GABA/benzodiazepine receptors in vitro.     

Sex Differences in the Effects of Acute Swim Stress on Binding to GABAA Receptors in Mouse Brain

Abstract: Acute swim stress (3-min swim at 32°C) in female, but not in male, mice results in substantial changes in the characteristics of GABA binding to membranes prepared from the forebrain. These changes were larger when measured in a relatively crude membrane preparation than in a well-washed membrane preparation commonly used in GABA binding assays, consistent with the loss of endogenous modulators of GABA binding in the latter preparation. These changes may be related to stress-induced alterations in part in the modulation of the characteristics of GABA binding by endogenous steroids, as the acute swim stress produced a larger increase in plasma corticosterone levels in female than in male mice.     

Photoaffinity Labelling of Benzodiazepine Receptors: Lack of Effect on Ligand Binding to the Nucleoside Transport System

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.     

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.     

Solubilisation of the γ-Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids

We have solubilised the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3-[(3-cholamidopropyl)dimethylammonio] 1-propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 +/- 0.2 nM compared to a KD of 2.3 +/- 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4 degrees C. This is contrary to the results obtained with a CHAPS-soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.     

Characterization with Antibodies of the γ-Aminobutyric AcidA/Benzodiazepine Receptor Complex During Development of the Rat Brain

The postnatal development of the gamma-aminobutyric acidA/benzodiazepine receptor (GABAR/BZDR) complex of the rat brain has been investigated using the monoclonal antibody 62-3G1 and the polyclonal rabbit antiserum A, specific for the 57,000 and 51,000 Mr receptor subunits, respectively. Both GABAR and BZDR binding activities co-precipitated during all postnatal ages. Adult rats showed a main 51,000 Mr[3H]flunitrazepam photoaffinity-labeled peptide, whereas newborn rats showed several photolabeled peptides of higher Mr. All the photolabeled peptides could be immunoprecipitated with each antibody regardless of the age of the rats. These results suggest that the physical coupling between the GABAR and the BZDR is already present in newborn animals and it is maintained afterwards during development. Glycosidase and peptidase treatments of the immunoprecipitated GABAR/BZDR complex indicated that all the [3H]flunitrazepam-photolabeled subunits are different peptides, although they seem to conserve a high degree of homology. In addition to the age-dependent heterogeneity, the results also suggest that for each age, there is heterogeneity in the subunit composition of the GABAR/BZDR complex.     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.     

Characterization of the Binding of [3H]Ro 5-4864, a Convulsant Benzodiazepine, to Guinea Pig Brain

The density of high affinity binding sites for [3H]4'-chlorodiazepam [( 3H]Ro 5-4864) in guinea pig cerebral cortex is significantly higher (3.8-fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5-4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5-4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.