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The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.

Summary Affinity column-purified GABA-benzodiazepine receptor proteins from human, cow, and rat brain were photoaffinity labeled with both [ 3 H]flunitrazepam and [ 3 H]muscimol and examined by gel electrophoresis in sodium dodecyl sulfate. Using high receptor protein concentrations (1 μM), the benzodiazepine ligand [ 3 H]flunitrazepam was incorporated covalently primarily into the expected 52 kiloDalton major subunit but also significantly into a second 57 kiloDalton peptide. Likewise the GABA ligand [ 3 H]muscimol photolabeled primarily the 57 kiloDalton peptide but also to some extent the 52 kiloDalton peptide. This cross-labeling suggests strongly that both major subunits carry binding sites for both GABA and benzodiazepine.

This paper describes a method used by EG&G Energy Measurements, Inc., to obtain high performance in a fast streak camera by using a magnification-equals-zero in the time direction. This method improves the dynamic range and temporal resolution and lowers chromatic aberrations. The results of the measurements on the streak tube and camera are provided herein.

The majority of inhibitory synaptic transmission in the central nervous system involves γ-aminobutyric acid (GABA) as the neurotransmitter (1). The signal transduction mechanism at the majority of GABA synapses involves a ligand-gated chloride channel; binding of GABA to its receptor increases postsynaptic membrane chloride conductance and inhibits the target cell (2). This GABA receptor, called GABAA, is defined pharmacologically by sensitivity to the agonist muscimol ana the antagonist bicuculline (3). At least one other type of GABA receptor exists, GABAB, defined as insensitive to bicuculline and sensitive to baclofen; GKBAB receptors are coupled to GTP-binding proteins for a variety of signal transduction mechanisms (4). GABAA receptor function is also modulated by at least three classes of centrally active drugs, the picrotoxin-like convulsants, that inhibit GABA function, and the benzodiazepines and the barbiturates, both of which enhance GABA function (5).

The function of the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered G AB A-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developed in vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.

The density of benzodiazepine/gamma-aminobutyric acid receptor binding sites was lower in the midbrain of seizure-susceptible gerbils compared to control seizure-resistant gerbils. Binding of [3H]diazepam to high-affinity brain-specific sites in membrane homogenates of gerbil brain showed a 20-30% lower binding in midbrain (but not other regions) in adult seizure-susceptible gerbils than in controls. This binding deficit was localized by tissue slice autoradiography with [3H]flunitrazepam to the substantia nigra and mesencephalic periaqueductal gray regions, while higher binding was observed in the interpeduncular nucleus. These differences were also seen in animals sacrificed immediately after a seizure. A parallel deficit of [3H]bicuculline methochloride binding to low-affinity gamma-aminobutyric acid receptors also was seen in the same midbrain regions. Scatchard plot analysis showed that the benzodiazepine binding deficit in the nigra was due to a lower number of binding sites with not significant difference in affinity. Lower [3H]flunitrazepam binding was likewise seen in younger animals (29% lower at 30 days of age, 38% at 60 days, and 21% at 90 days), indicating that the midbrain receptor deficit is present in the seizure-susceptible gerbil prior to the age of onset of seizures at 50-100 days. Therefore, these changes are not likely to result from seizures but reflect genetically determined biochemical differences that could play a role in the expression of seizure susceptibility. The deficit in midbrain benzodiazepine/gamma-aminobutyric acid receptors in the seizure-susceptible gerbil would be consistent with the hypothesis that a deficit of gamma-aminobutyric acid-mediated inhibition might contribute to some kinds of epilepsy.

A radioimmune assay for microtubule protein, tubulin, is described, in which unknown amounts of native or denatured tubulin can be quantitated by the ability to compete with pure [125I]tubulin for rabbit antibodies produced against purified bovine brain tubulin. The assay is used to demonstrate that crude extracts of mouse brain contain negligible amounts of 30–36S tubulin oligomers under conditions where purified tubulin forms substantial amounts of such structures. Also, the particulate fraction of osmotically shocked and sonicated brain synaptosomes contains negligible tubulin antigenic activity. By contrast, soluble extracts of soybean, especially rapidly dividing regions of the plant, were found to contain significant amounts of cross-reacting material, providing further evidence for the conservative evolutionary nature of this ubiquitous and important protein.

Summary Binding activity of the radioactive cage convulsant [35S] t -butyl-bicyclophosphorothionate was solubilized from rat brain membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio] propanesulfonate. Binding (KD = 26 nM, Bmax = 0.4 pmol/mg protein) was inhibited by picrotoxin and related convulsants and by barbiturates and related depressants that interact with γ-aminobutyric acid and benzodiazepine receptors via the picrotoxinin binding site. The convulsant/ barbiturate binding activity chromatographed on gel filtration as a single peak coinciding with the benzodiazepine/γ-aminobutyric acid receptor protein complex.

The novel convulsant amidine steroid RU5135 inhibited gamma-aminobutyric acid receptor binding in membranes from seven regions of rat brain (IC50 = 11 +/- 2 nM against [3H]muscimol and 0.8 +/- 0.2 nM against [3H]bicuculline methochloride), apparently lowering the number of binding sites labeled with gamma-aminobutyric acid receptor agonists or antagonists. The steroid reversed the enhancement of benzodiazepine receptor binding by gamma-aminobutyric acid, pentobarbital, and etazolate, but did not inhibit the binding of the convulsant [35S]t-butyl bicylophosphorothionate. Thus, RU5135 shows very potent in vitro actions more resembling those of the gamma-aminobutyric acid site antagonist, bicuculline, than the chloride channel antagonist, picrotoxin.

γ-Aminobutyric acid is the major inhibitory neurotransmitter in the central nervous system and is widely utilized in virtually all anatomic regions of the brain and spinal cord [1,2]. In recent years, pharmacological evidence at all levels (organismal, tissue, cellular, and molecular) have implicated GABA in a variety of human clinical problems and, correspondingly, in the action of numerous drugs showing general excitatory or depressant action on the central nervous system [3–5]. These include convulsants like picrotoxin [6], bicuculline [7], cage convulsants [8], pentylenetetrazol [9], and convulsant benzodiazepines [10, 11], as well as depressants like benzodiazepines (used for anxiolytic, anticonvulsant, sedative-hypnotic, or muscle relaxant activity [12], barbiturates (used today as anticonvulsants and general anesthetics, and formerly as sedative-hypnotics [13]), other general anesthetics [14, 15], including ethanol [16, 17], and a variety of related substances [18]. Because of the widespread function of GABA in the brain, interactions with virtually all neuropharmacological agents and other neurotransmitters have been described. However, increasing bodies of evidence suggest that the drugs mentioned above act by modifying the postsynaptic response to GABA directly at the GABA receptor-chloride ion channel complex [19,20].

GABA (γ-aminobutyric acid) is now established as the major neurotransmitter in mammalian brain, and mediates its effects via the GABA receptor and an associated chloride ion channel (1]. Further accumulated evidence suggests that some GABA receptors possess modulatory sites for certain centrally active compounds, the benzodiazepines and the barbiturates [1].

Several types of cage compounds are highly toxic to mammals. These include tetramethylene disulfotetramine, alkyl bicyclophosphate esters (4-alkyl-1-phospha-2, 6,7-trioxabicyclo [2.2.2] octane-1-oxides), ortho carboxylic acids of related structure (1-substituted-4-alkyl-2,6,7-trioxabicyclo [2.2.2] octanes), and silicon-nitrogen (silatrane) analogues (1-substituted-2,8,9-trioxa-5-aza-1-silabicyclo [3.3.3] undecanes). These compounds have convulsant activity resembling the action of picrotoxin, a synaptic antagonist of the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). In this study, numerous examples of these cage compounds have been found to have no effect on GABA receptor binding sites in mammalian brain; rather, they inhibit potently the binding of dihydropicrotoxinin to membrane sites related to the chloride ion channels which are regulated by GABA receptors.

Bicuculline and 3 chemical derivatives were assayed on a variety of biological systems. Consistent with reports of studies on other animals, some of these compounds caused convulsions in insects and blocked inhibitory postsynaptic potentials in insect muscle. They all potently inhibited mouse brain acetylcholinesterase. Bicuculline and its analogs inhibited the binding of GABA in vitro to sites in crayfish muscle membranes which have properties of receptor sites; this site of action could explain the activity of bicuculline at arthropod neuromuscular junctions. These compounds, at high concentrations (over 100 muM), also inhibited GABA uptake by mouse brain homogenates at 0 degrees C apparently non-competitively. Bicucine methyl ester inhibited GABA transport by brain at 37 degrees C, consistent with non-specific membrane effects at high concentrations of drug. These and other observations cast doubt upon the specificity of bicuculline-like compounds for action on GABA synapses, especially for in vitro studies at high drug concentrations (over 10 muM). The neuroactivity of low doses of bicuculline is apparently not explained by these in vitro effects, and could very well be due to inhibition of GABA synapses at either receptor or ionophore sites. At physiological conditions of pH and temperature, bicuculline is hydrolyzed at its lactone moiety to the less active compound bicucine; this could lead to underestimates of the biological activity of bicuculline. More stable analogs studied so far are not more potent, however.

1. Acetylcholine.- 2. Glutamate.- 3. GABA.- 4. Amine Transmitters and their Associated Second Messenger Systems.- 5. Invertebrate Neuropeptides.- 6. Neuronal Cultures as Experimental Systems.- 7. Neurotoxins.

The Mongolian gerbil (Meriones unguiculatus) was first described to the western scientific community by Milne-Edwards (1867). The species was introduced to the United States in 1954 by Schwentker (see Schwentker, 1972) and these animals formed the basis of the colony developed by Tumblebrook Farms, West Brookfield, Massachusetts, currently the major breeders and suppliers. Gerbils were first bred at UCLA by Rich in 1967 (Rich, 1968). Abnormal behaviors were soon noted in some of the animals and were recognized as epileptiform in nature by Loskota, who decided to study the gerbil as a model of the epilepsies; this research subsequently constituted the basis of his dissertation for the degree of doctor of philosophy at UCLA (Loskota, 1974).

The specific binding of [3H] alpha-dihydropicrotoxinin to rat brain membranes was inhibited competitively and potently (IC50 congruent to 100 nM) by a convulsant benzodiazepine drug, RO5-3663. This compound did not inhibit high affinity flunitrazepam binding to the same tissue under similar conditions, and its reported pharmacological activity as an antagonist of GABAergic synaptic transmission, which resembles that of picrotoxinin, appears to involve the picrotoxinin binding sites. Other benzodiazepines such as diazepam, in micromolar concentrations, inhibited picrotoxinin binding in a stereospecific and chemically specific manner. However, the order of potency of a series of depressant benzodiazepines did not correlate well with pharmacological activities nor with reported activities for displacement of high affinity benzodiazepine 'receptor' binding sites (although heterogeneity of both picrotoxinin and benzodiazepine binding site populations may make difficult such comparisons). A comparison of benzodiazepine-displaceable benzodiazepine binding and benzodiazepine-displaceable picrotoxinin binding for different brain regions and subcellular fractions revealed a very similar though not identical distribution of these two classes of drug receptor, again suggesting that the two are not identical. Both classes of drug binding site also showed a very similar distribution to sodium-independent GABA receptor binding sites, which is consistent with other evidence that at least part of these 3 receptor types may be found at least sometimes coupled together in the postsynaptic membrane GABA receptor-ionophore complex.

The binding of [(3)H]muscimol to membrane fractions from central nervous tissue from fly (Musca domestica) and locust (Schistocerca gregaria) was measured. Specific saturable binding was observed with K(d)s of 10 nM (locust) and 40 nM (fly) and B(max) values of 20 fmol/mg protein (fly) and 60 fmol/mg protein (locust). Binding was Na(+)-independent and in locust tissue an additional Na(+)-dependent binding component was observed. Pharmacological properties of the binding sites in both species are consistent with a GABA receptor site rather than an uptake/transport site. We conclude that insect central nervous tissue contains receptors that show some similarities to the GABA receptor complex of mammalian brain.

Barbiturate drugs of diverse chemical structure inhibited the binding of [ 3 H] α-dihydropicrotoxinin to rat brain membranes. This biologically active analoque of picrotoxin labels membrane sites related to the convulsant action of these drugs in inhibiting GABA postsynaptic receptor-ionophore function at a site distinct from the GABA receptor. Depressant barbiturates such as pentobarbital inhibited dihydropicrotoxinin binding competitively at therapeutic concentrations (IC 50 = 50 μ M) whereas the drug does not alter GABA receptors, uptake, or release at this concentration. Antiepileptics such as phenobarbital (IC 50 =400 μ M), were weaker inhibitors of binding. Convulsant barbiturates, however, such as dimethylbutylbarbiturate (IC 50 =0.05 μ M) and cyclohexylidene-ethyl barbiturate (IC 50 =0.7 μ M), were potent inhibitors. The displacement of radioactive dihydropicrotoxinin binding by the convulsant barbiturates had different slopes and Hill numnbers (0.4) compared to displacement by depressant barbiturates and picrotoxinin itself (Hill numbers = 1.0), indicating heterogeneity of binding sites or negative cooperativity. These potent intractions of barbiturates with dihydropicrotoxinin binding sites are consistent with neurophysiological evidence that depressant or convulsant action of barbiturates may involve modulation of CNS inhibitory synaptic transmission at the level of the postsynaptic GABA receptor-ionophores.

The post synaptic receptor for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is a complex protein containing a chloride channel and modulatory sites for two classes of drugs, the benzodiazepines (BZ) and convulsants/barbiturates. Barbiturates which enhance GABA-activated chloride permeability at the tissue and cellular level also are able to enhance benzodiazepine and GABA receptor binding at the molecular level. Convulsant/barbiturate sites can be assayed directly with [ 35 S]t-butyl bicyclophosphorothionate (TBPS). This activity was solubilized with the detergent CHAPS and shown to co-migrate with BZ and GABA receptor binding activity during protein fractionation as a single protein complex, which could be purified intact by over 1000-fold on benzodiazepine affinity columns.

GABA receptor binding curves were measured in well-washed membrane homogenates from eleven regions of normal human brain and four regions from brains of Huntington disease patients. Computer analysis suggested two populations of receptor sites of different affinity (KD = 10 nM and 240 nM) present in all brain regions but in variable quantity (cerebellar cortex and cerebral cortex greater than basal ganglia greater than deeper brain structures). The number of GABA receptor sites in the caudate-putamen region of Huntington brain was less than normal, but the number of sites was increased in Huntington disease substantia nigra. No differences from normal were found in cerebellar cortex or frontal cortex from Huntington disease, and no significant changes in binding affinity were observed for any of the four regions tested under the conditions used.