Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

Peripheral-type benzodiazepine-binding sites in platelets of schizophrenics with and without tardive dyskinesia

The density of peripheral-type benzodiazepine (BZ)-binding sites was studied in platelets of 10 medicated chronic schizophrenics with tardive dyskinesia (TD), 10 medicated chronic schizophrenics without TD, 7 drug-free schizophrenics, and 10 normal controls. The age range of the study population was 36-60 years. Age and sex distribution were similar in all 4 groups. The unmedicated schizophrenics did not differ in their maximal binding capacity from the healthy controls. A significant decrease in the density of peripheral-type BZ-binding sites in platelets was observed in treated schizophrenics both with and without TD in comparison to controls and untreated schizophrenics. The reduction in [3H]PK 11195 binding was more pronounced in TD patients (31.3% of controls) than in patients without TD (21.1% of controls). However, this parameter failed to discriminate statistically between TD and non-TD medicated schizophrenics.     

Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain

The effects of acute and chronic administration of a subconvulsive dose of picrotoxin on t-[35S]butylbicyclophosphorothionate ([3S]TBPS), [3H]muscimol, and [3H]flunitrazepam binding characteristics in various regions and on the convulsant potency of picrotoxin in Sprague-Dawley rats were examined. Acute administration of a subconvulsive dose of picrotoxin (3 mg/kg, i.p.) significantly increased [35S]TBPS and [3H]muscimol binding in cerebellum (CB) with no change in frontal cortex (FC). In rats treated chronically with picrotoxin (3 mg/kg, i.p., daily for 10 days), the Bmax of [35S]TBPS binding site was significantly decreased in the FC, striatum (ST), and CB with no change in KD values. Neither [3H]muscimol binding in the FC and CB nor [3H]flunitrazepam binding in the FC was affected in these rats. In addition, the potency of pentobarbital to inhibit [35S]TBPS binding in vitro was not altered following acute or chronic treatment of picrotoxin. Chronic administration of picrotoxin did not affect convulsive ED50 or LD50 of picrotoxin; however, it delayed the onset of convulsions and increased the time to death. These results suggest that treatment with picrotoxin at a subconvulsive dose for 10 days causes down-regulation of [35S]TBPS binding sites and that this down-regulation might be related, at least in part, to the decreased extent of convulsant potency of picrotoxin. In addition, the results indicate possible interaction between convulsant binding sites and GABAA receptor sites in the CB following picrotoxin treatment.     

Normal coupling of brain benzodiazepine and neurosteroid modulatory sites on the GABA-A receptor complex in human hepatic encephalopathy

To assess the possible implication of the allosteric coupling of different modulatory sites at the GABA-A receptor complex in hepatic encephalopathy (HE), we investigated in autopsied frontal cortex of six cirrhotic patients and six appropriately-matched controls, the modulatory effects of the benzodiazepine site agonist flunitrazepam on the binding of [3H]muscimol and the effect of the neurosteroid site agonist allopregnanolone (5alpha-pregnan-3alpha-ol-20-one) on the binding of [3H]muscimol and [3H]flunitrazepam. There were no significant differences in either the magnitude E(max): 11.5+/-1.1% (controls) versus 10.2+/-2.2% (HE patients) or the efficacy EC(50): 20.2+/-5.5 nM (controls) versus 17.7+/-6.2 nM (HE patients) of flunitrazepam modulation of [3H]muscimol binding. Allopregnanolone also showed modulation of both sites to a comparable extent in brain tissue from cirrhotic patients and controls E(max): [3H]muscimol, 15.1+/-2.8% (controls) versus 13.8+/-1.9% (HE patients); [3H]flunitrazepam, 17.9+/-2.3% (controls) versus 19.1+/-2.3% (HE patients), EC(50): [3H]muscimol, 386.5+/-25.8 nM (controls) versus 373.8+/-13.1 nM (HE patients); [3H]flunitrazepam, 49.8+/-22.9 nM (controls) versus 55.5+/-14.0 nM (HE patients). These findings demonstrate unequivocally that the GABA-A sites and their benzodiazepine and neurosteroid modulatory sites manifest normal allosteric coupling in brain in human HE. Therefore, if increased "GABAergic tone" is implicated in the pathophysiology of HE, this must be the consequence of increased brain concentrations of endogenous benzodiazepine and/or neurosteroid ligands for components of the GABA-A receptor complex rather than alterations of the receptor proteins themselves.     

Bicuculline Up-Regulation of GABAA Receptors in Rat Brain

Effects of acute and subacute administration of bicuculline on [3H]muscimol, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to various brain regions were studied in Sprague-Dawley rats. Acute administration of bicuculline affected neither the KD nor the Bmax of the three receptor sites. In rats treated subacutely with bicuculline (2 mg/kg, i.p., daily for 10 days), [3H]muscimol binding was increased in the frontal cortex, cerebellum, striatum, and substantia nigra. Scatchard analysis revealed that subacute treatment of rats with bicuculline resulted in a significantly lower KD of high-affinity sites in the striatum and in a significantly lower KD of high- and low-affinity sites in the frontal cortex. In the cerebellum, two binding sites were apparent in controls and acutely treated animals; however, only the high-affinity site was defined in subacutely treated animals, with an increase in the Bmax value. Triton X-100 treatment of frontal cortical membranes eliminated the difference in [3H]muscimol binding between control and subacute bicuculline treatments. On the other hand, [3H]muscimol binding was significantly increased in the cerebellum from bicuculline-treated animals even after Triton X-100 treatment. The apparent Ki of bicuculline for the GABAA receptor was also decreased in the frontal cortex and the striatum following the treatment. However, subacute administration of bicuculline affected neither the KD nor the Bmax of [3H]flunitrazepam and [35S]TBPS binding in the frontal cortex and the cerebellum. These results suggest that GABAA receptors are up-regulated after subacute administration of bicuculline, with no change in benzodiazepine and picrotoxin binding sites.     

[3H]muscimol and [3H]flunitrazepam binding sites in the developing cerebellum of mice treated with methylazoxymethanol at different postnatal ages

Two models of perturbed cerebellar ontogenesis were obtained by a single administration of methylazoxymethanol (MAM), a potent antimitotic agent, to mouse pups either on the day of birth (MAM₀ mice) or at postnatal day 5 (MAM₅ mice). The alterations of the cerebellar GABAergic system were studied by measuring glutamic acid decarboxylase activity, [³H]muscimol binding sites, which are known to be concentrated in the GABA_A receptors in the internal granular layer, and [³H]flunitrazepam binding sites, which are more abundant in the molecular layer. The primary target of the antimitotic agent are the precursors of the glutamatergic and GABAceptive granule cells. In both models GABAergic structures, as revealed by GAD activity measurements, appear to be relatively spared, and recovery of granule cell numbers occurs during development in MAM₅ mice. In MAM treated mice the number of [³H]muscimol binding sites (on a per cerebellum basis) decrease as the number of granule cells decrease, although some recovery occurred in MAM₅ mice, but not in MAM₀ mice. In MAM₅ mice, [³H]flunitrazepam binding sites (on a per cerebellum basis) were relatively unaffected, while they were decreased significantly, but to a lesser extent than [³H]muscimol binding sites, in MAM₀ animals. The more significant reduction of granule cell numbers and the cytoarchitectural disruption resultant from the more precocious application of the antimitotic appear responsible for the significant alteration and lack of recovery in MAM₀ mice.     

Glycoprotein as a Constituent of Purified γ-Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

γ-Aminobutyric Acid and Benzodiazepine Receptor Changes Induced by Unilateral 6-Hydroxydopamine Lesions of the Medial Forebrain Bundle

Quantitative autoradiography was used to ascertain alterations in [3H]muscimol, [3H]flunitrazepam (FLU), [3H]naloxone, [3H]D-alanine-D-leucine-enkephalin (DADL), and [3H]spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, [3H]spiroperidol binding increased 33% in striatum. At 5 months, [3H]spiroperidol was only nonsignificantly increased above control. At 1 week, [3H]muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, [3H]muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in [3H]muscimol binding persisted at 5 months. [3H]FLU binding was altered in the same direction as [3H]muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in [3H]naloxone and [3H]DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr.     

Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

Modification of the GABA/benzodiazepine receptor with the arginine reagent, 2,3-butanedione

Treatment of either crude or purified preparations of the gamma-aminobutyrate (GABA)/benzodiazepine receptor complex with arginine-specific reagents resulted in a time- and concentration-dependent loss of [3H]muscimol binding activity. Following exposure to either 2,3-butanedione or phenylglyoxal (less than or equal to 20 mM), [3H]muscimol binding was inhibited by up to 80%. [3H]Flunitrazepam binding was much less sensitive to the effects of the reagents. Scatchard analysis of the binding data indicated that treatment with butanedione resulted in a loss of [3H]muscimol binding sites with little effect on binding affinity. Considerable protection against inactivation was provided by arginine and by the endogenous receptor ligand, GABA. These results indicate that arginine residues play a critical role in maintaining the GABA receptor in a conformation capable of ligand binding, possibly by participating in the binding site through interaction with the carboxylate moiety of GABA.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).     

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.     

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [³H]flunitrazepam, [³H]GABA or [³H]muscimol to extensively washed synaptic membranes. Neither the affinity (K_d) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [³H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [³H]flunitrazepam or [³H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA—benzodiazepine receptor ionophore-complex in the brain.     

Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia.

Binding studies with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), a specific serotonin1A (5-HT1A) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. All the patients and controls were of the Japanese race. In the controls, representative Scatchard plots for the specific [3H]8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site (Kd value = 5.7 and 5.9 nM, Bmax value = 80.1 and 101.0 fmol/mg protein, respectively). The [3H]8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin (IC50 = 6.3 x 10(-9) M) and 5-HT1A agonists (IC50 = 5.0 x 10(-9) - 2.3 x 10(-7) M), while other neurotransmitters, 5-HT2 and 5-HT3 related compounds did not inhibit the binding (IC50 greater than 10(-5) M). The bindings were decreased in the presence of 0.1mM GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific [3H]8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls. Scatchard analysis showed that this increased binding reflects changes in the number of sites but not in the affinity. The effect of 0.1mM GppNHp on the binding to prefrontal cortex was observed in both controls and schizophrenic patients. The bindings were significantly greater in the schizophrenic patients than in controls, in the presence of 0.1mM GppNHp. Our findings suggest that there are GTP-sensitive 5-HT1A sites in the human brain and that selective increases in GTP-sensitive 5-HT1A sites in the prefrontal and temporal cortices of schizophrenics relate to the pathophysiology of schizophrenia.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

Alteration of dopamine receptors in the caudate nucleus and the putamen in schizophrenic brain

Neuroleptic binding to human caudate and putamen was investigated in seven patients with schizophrenia and compared to matched normal controls. [3H]-spiperone was used as a ligand for the binding studies and previous drug treatment was recorded. There was a statistically significant increase in maximal specific binding and in dissociation constants for [3H]-spiperone in the brains of schizophrenics in both brain regions studied. Long term as well as recent neuroleptic treatment both appeared to be associated with increases of Bmax and Kd of [3H]-spiperone.     

Isolation and characterization of endogenous modulators for GABA system

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with eitherl-glutamate or GABA. However, others are clearly separated froml-glutamate or GABA. EGIs were found to be low MW (<1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (<1,800 dalton) neutral or positively charged substances. MBIs had no effect on [³H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.Special issue dedicated to Dr. Frederick E. Samson