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Increase in the Bmax of Y-Aminobutyric Acid-A Recognition Sites in Brain Regions of Mice Receiving Diazepam

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Increase in the Bmax of Y-Aminobutyric Acid-A Recognition Sites in Brain Regions of Mice Receiving Diazepam Proc. Nati. Acad. Sci. USA Vol. 81, pp. 2247-2251, April 1984 Neurobiology Increase in the Bmax of y-aminobutyric acid-A recognition sites in brain regions of mice receiving diazepam (muscimol/-aminobutyric acid receptors/benzodiazepine receptors/locomotor activity) P. FERRERO, A. GuIDOTTI, AND E. COSTA* Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, DC 20032 Contributed by E. Costa, December 12, 1983 ABSTRACT y-Aminobutyric acid (GABA) receptors were MATERIALS AND METHODS characterized in vivo by studying ex vivo the binding of [3H]Muscimol (New England Nuclear) labeled on the meth- [3H]muscimol to cerebellum, cortex, hippocampus, and cor- ylene side-chain (specific activity, 29.4 Ci/mmol; 1 Ci = 37 pus striatum of mice receiving intravenous injections of tracer GBq) was injected into the tail vein of female mice (20-25 g). doses of high-specific-activity (-30 Ci/mmol) [3H]muscimol. Routinely, the dose of [3H]muscimol injected was 5 uCi per This ligand binds with high affinity (apparent Kd, 2-3 x 10-9 20 g of body weight in 0.2 ml of saline. This radiolabeled M) to a single population of binding sites (apparent Bmax, 250- ligand dose was administered either alone or with various 180 fmol per 10 mg of protein). Pharmacological studies using doses of unlabeled muscimol. In some experiments, drugs that selectively bind to GABAA or GABAB receptors [3H]muscimol was injected into cerebral ventricles through a suggest that [3H]muscimol specifically labels a GABAA recog- polyethylene cannula chronically implanted (13). The dissec- nition site. Moreover, diazepam (1.5 ,umol/kg, i.p.) increases tion of the different brain areas (cerebellum, cortex, stria- the Bmax but fails to change the affinity of [3Hlmuscimol bind- tum, and hippocampus) was carried out on a chilled (00C) ing to different brain areas. This diazepam-elicited increase in glass plate as described by Glowinski and Iversen (14) for the Bmax is blocked in mice receiving the diazepam antagonist Ro rat brain. The cortex, portion B of Glowinski and Iversen 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo4H-imidazo- (14), was dissected by using the coronal plane passing [1,5a]-[1,4jbenzodiazepine-3-carboxylate). Since the diazepam- through the anterior commissura as anterior reference point. induced increase of [ H]muscimol binding is paralleled by a Authentic [3H]muscimol was extracted from brain tissue significant potentiation of the inhibitory effect of muscimol on as described (13). Various brain areas (10-100 mg) were ho- locomotor activity, it is proposed that the facilitatory action on mogenized in 2.5 ml of 0.4 M HCl04 immediately after dis- GABAergic transmission elicited in vivo by diazepam is medi- section. The acid supernatant (2 ml) from centrifugation at ated by an increase in the Bmax of the binding sites of GABAA 30,000 x g for 30 min was applied to an AG 50 x 8 column receptors. (3H, 200-400 mesh, 0.5 x 5 cm high). The eluate was collect- ed and the column was washed first with 2 ml of HO and The in vivo potentiation of y-aminobutyric acid (GABA)ergic then with 7 ml of 2 M NH40H. [3H]Muscimol was eluted in responses elicited by doses of benzodiazepines that in the the NH40H fraction and migrated as authentic muscimol on absence of GABA are devoid of action has suggested the Silica gel G thin-layer chromatography using ethylacetate/ widely accepted view that almost every action of benzodia- isopropanol/ammonium hydroxide (9:7:4) or n-butanol/acetic zepines is mediated by a facilitation of GABAergic transmis- acid/water (25:4:10) as solvents. The recovery of [3H]musci- sion (1, 2). Experiments with crude synaptic membrane mol throughout the entire procedure was -75%. In prelimi- preparations have given support to this view by showing that nary experiments, we have estimated the biological half-life many anxiolytic benzodiazepines increase the Bmax of of [3H]muscimol in mouse brain. Various brain areas were GABA recognition sites (3-6). However, it remains to be dissected from mice receiving intravenously 0.0085 /imol of ascertained whether in vitro interactions between GABA [3H]muscimol per kg of body weight. In all the brain areas recognition sites and benzodiazepines (3, 7) or,-carboline-3- studied and between 10 and 60 min after the injection of carboxylic esters (8, 9) are also operative in vivo. To verify [3H]muscimol, authentic radioactive muscimol represented whether such an extrapolation is possible, we have com- between 15% and 20% of the total radioactivity. The concen- pared ex vivo binding of [3H]muscimol to brain structures of tration of [3H]muscimol was the highest at 10 min and rapid- mice receiving saline or diazepam. This experimental model ly decreased to 40% of the peak value between 40 and 60 has given us an opportunity to verify whether the potency of min. In the various brain areas, the [3H]muscimol content various GABAergic drugs relates to the degree of GABA was decreased by =50% when the specific activity of the recognition-site saturation. Such a correlation had been tracer dose of [3H]muscimol was decreased to 0.4% of the shown for ligands of opiate (10), dopamine (11), and benzodi- original value. The greatest relative decrease was observed azepine (12) recognition sites but not for ligands of GABA 10 min after the injection. Hence, in the present experiments receptors. this time interval was used to study the specific binding of We report here that [3H]muscimol injected in the mouse [3H]muscimol. When [3H]muscimol was injected into the ce- binds to high-affinity GABA recognition sites present in var- rebral ventricles, >90% of the total radioactivity extracted ious brain structures. We also show that in mice receiving from brain was recovered as authentic muscimol 10 min after diazepam, the Bmax of [3H]muscimol binding increases. the injection. In these experiments, the proteins were deter- These results suggest that with the use of an appropriate ra- mined by the method of Lowry et al. (15). dioligand one could study in man the GABA receptor func- GABA Measurements. Brain areas, rapidly dissected on a tion in vivo using positron emission tomography. chilled glass, were homogenized in 0.4 M HC104/50 nmol of citrulline per ml as internal standard and centrifuged at The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: GABA, y-aminobutyric acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2247 Downloaded by guest on October 1, 2021 2248 Neurobiology: Ferrero et aLPProc. Nad Acad Sci. USA 81 (1984) 33,000 x g for 15 min. The supernatant was injected into a jections of [3H]muscimol at 8.5 nmol/kg either alone or in HPLC system equipped with a 3.1 mm x 25 cm BioRad combination with increasing concentrations of unlabeled Aminex A-9 cation exchange resin column maintained at drug. Two main compartments of [3H]muscimol can be iden- 550C. Amino acids were eluted from the column by a step tified: a displaceable compartment, which declines linearly gradient buffer as described by Schmid et al. (16). The col- with the log of the [3H]muscimol specific activity, and a non- umn effluent was mixed with o-phthalaldehyde to derivatize displaceable compartment (-50% of the total cerebellar the primary amino groups for fluorimetric quantification of [3H]muscimol), which reaches a plateau level in mice receiv- GABA (see ref. 16 for details). ing muscimol doses of 5-50 Amol/kg. Assuming that the dis- Behavioral Tests. Mice were housed for 4 days or longer placeable compartment represents muscimol bound to high- with constant temperature and humidity in our facilities with affinity specific recognition sites and the nondisplaceable 12-hr light cycles (6 a.m. to 6 p.m.). All the experiments compartment represents the nonspecifically bound and/or were done between 2 and 5 p.m. free muscimol, the net amount of muscimol bound to high- Administration of isoniazid (350 mg per kg, s.c.) induces, affinity recognition sites (curve B) can be calculated by sub- within 30-40 min, tonic-clonic seizures characterized by tracting from the [3H]muscimol content found in mice re- prolonged periods of hindlimb extension. The time interval ceiving this drug the [3H]muscimol contained in the brain of between the isoniazid injection and the onset of tonic-clonic mice receiving a high dose of nonlabeled muscimol. Results seizures was taken as the "convulsion latency time." Musci- similar to those obtained in Fig. 1 were obtained when unla- mol or saline were injected intravenously 20 min after isonia- beled muscimol was injected intravenously 10 min before the zid. tracer compound. Locomotor activity was tested in a sound-proof room by To analyze whether the binding of [3H]muscimol to cere- placing each mouse in a clear plastic box on an Electron Mo- bellum follows the mass-action law, saturation curves were tility Meter, 40 FC (Motron Products, Stockholm, Sweden). constructed by intravenously injecting increasing concentra- The motility meter includes infrared light beams shining tions of 3H-labeled ligand (0.002-0.5 ,umol/kg). As shown in from above and 40 detector cells underneath the box floor. Fig. 2, the binding of [3H]muscimol is saturable for muscimol Mice locomotion was recorded as light-beam interruptions doses >0.1 Amol/kg. The apparent number of muscimol by a digital meter. The counts were added for time intervals binding sites, calculated by analyzing the data of Fig.
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