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 (/-aminobutyric acid receptors/ 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 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 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 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 (350 mg per kg, s.c.) induces, affinity recognition sites (curve B) can be calculated by sub- within 30-40 min, tonic-clonic 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. 2 using of 2.5 min for a cumulative time period of 10 min. Naive a Lineweaver-Burk plot, is 230 fmol per 10 mg of protein. mice, receiving either vehicle or drugs (diazepam or musci- The data of the saturation studies fit into the Hill plot equa- mol), were used for this test. tion yielding a straight line with a slope of 0.98 and a Hill binding constant of 0.08 ,umol/kg. Since in mice receiving RESULTS [3H]muscimol at 0.08 ,mol/kg the drug concentration in cer- Ex Vivo Measurements of [3lH]Muscimol Binding to Mouse ebellum is 2-3 nM, it can be extrapolated that in these ex Brain Regions. The [3H]muscimol binds in a saturable man- vivo studies muscimol binding has a Kd value of about 2-3 x ner to cortex, striatum, hippocampus, and cerebellum of 10-9 M. mice receiving this label intravenously. Fig. 1 depicts the Saturable high-affinity binding curves for [3H]muscimol cerebellar content of [3H]muscimol after the intravenous in- similar to the one depicted in Fig. 2 for cerebellum have been obtained in cortex (Bmax, 180 fmol per 10 mg of protein; Hill constant, 0.06 ,mol/kg), hippocampus (Bmax, 240; Hill con-

c'-. 10 - O.o

._ A E 4o0D -: 0 0^ .90 c)- .0 UE xDU -. = ° ._ 0) 4.4 t_ . 0 _ S.v =

C4

0 x 0.01 0.1 0.5 Muscimol, ,mol/kg 2- FIG. 2. Potentiation by diazepam of [3H]muscimol binding to cerebellum. Saturation curves were obtained by i.v. injections of

I . I I . increasing concentrations of radioligand. The doses of [3H]musci- 0.01 0.1 0.5 5 50 mol between 2 and 8.5 nmol/kg were obtained by increasing the Muscimol, ,mol/kg amount of [3H]muscimol injected. Thus, in this dose range the spe- cific activity was kept constant. For the [3H]muscimol doses be- FIG. 1. Dose-dependent displacement in mouse cerebellum of tween 10 and 500 nmol/kg, a constant amount of [3H]muscimol (8.5 [3H]muscimol by unlabeled muscimol. Mice received [3H]muscimol nmol/kg) was mixed with various amounts of unlabeled muscimol. at 8.5 nmol/kg together with increasing doses (i.v.) of unlabeled In this dose range for each point the amount of [3H]muscimol specif- muscimol. Curve A represents the concentration of [3H]muscimol in ically bound to cerebellum was calculated by subtracting from the cerebellum determined 10 min after the injection. Broken line de- total [3H]muscimol the amount nondisplaceable by an excess (5 notes the compartment of [3H]muscimol nondisplaceable by unla- ,umol) of unlabeled muscimol. The animals received vehicle (0) or beled muscimol. Curve B, which represents the displaceable diazepam (o) (1.5 ,mol/kg, i.p.) 5 min before the injection of the [3H]muscimol compartment, was calculated by subtracting from the radioligand. Muscimol content in tissue was determined 10 min after total concentration of [3H]muscimol (curve A) the concentration at- the injection. Each point is the mean + SEM of at least 10 mice. The tributable to the nondisplaceable compartment. Each point of curve amount of nondisplaceable muscimol was the same in control and A is the mean ± SEM of at least 10 mice. diazepam-treated mice. Downloaded by guest on October 1, 2021 Biochemistry: Stem and Palmer Proc. Natl. Acad. Sci. USA 81 (1984) 1949

1 2 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

11.5 kb-_ 105

67 m 5.5

27 0 _ *

SPINACH BAM 115 Smt 105 Smt 67 Smt 55

FIG. 5. Hybridizations of nick-translated spinach mtDNA and ctDNA clones to BamHI-digested spinach ctDNA (lanes 1), BamHI-digested spinach Bam 11.5 (lanes 2), Sal I-digested spinach mtDNA (lanes 3), Sal I-digested Smt 10.5 (lanes 4), Sal I-digested Smt 6.7 (lanes 5), and Sal I- digested Smt 5.5 (lanes 6); Smt, spinach mtDNA clone, followed by the size of the insert in kb. Hybridization probes are given below each panel. In each case, with the exception of Smt 5.5, a fragment of about 20 kb (-.) is identified in the mtDNA track. This fragment is a Sal I fragment from contaminating ctDNA (see Fig. 4 and text). Smt 6.7 appears to hybridize to a ctDNA fragment other than Bam 11.5. This is consistent with the hybridization of MB 18.8 to a 6.7-kb spinach mtDNA fragment (Fig. 3), which we have confirmed by reciprocal hybridiza- tions between Smt 6.7 and MB 18.8 (data not shown). In addition, the relatively strong hybridization of Smt 6.7 to the 20-kb region of spinach mtDNA reflects not only ctDNA-contamination but also sequence homology between Smt 6.7 and a bonafide spinach mtDNA fragment of 19.5 kb (unpublished data). The 2.7-kb restriction fragment seen in lanes 2, 4, 5, and 6 is the cloning vector pUC 8 (11).

these mitochondrially located transferred sequences should ization of corn mtDNA to ctDNA rbcL sequences (Fig. 4) be significantly more closely related to ctDNA from one spe- reflects a corn mtDNA rbcL sequence that is virtually identi- cies than from the other. Experimental results compatible cal to its corn ctDNA homolog (21). with this hypothesis were obtained when mung bean and Mechanism of Interorganeliar Sequence Transfer. There spinach ctDNA clones (MB 11.1 and Bam 11.5, respective- are two types of mechanism that could account for random ly), which contain similar sequences (3), were hybridized to and widespread sequence transfer between cytoplasmic or- mung bean and spinach mitochondrial DNAs. MB 11.1 hy- ganelles. One type would require direct physical contact. bridized very strongly to a 5.5-kb mung bean mtDNA restric- Membrane continuities and other associations between chlo- tion fragment and less intensely to 5.5-, 6.7-, and 10.5-kb roplast and have been reported for several spinach mtDNA fragments (Fig. 3). Reciprocal hybridization species, including barley (22), corn (23), Hyptis suaveolens intensities were obtained with Bamr 11.5 (Fig. 4). Relative to (23), Pteris vittata (24), tobacco (25), Panicum schenckii their hybridization with MB 11.1, all three spinach mtDNA (26), and Euglena (27). Moreover, Wildman et al. (28) ob- fragments hybridized more strongly to Bam 11.5 than did the served various physical interactions between the two organ- mung bean 5.5-kb mtDNA fragment. Additionally, whereas elles in cinematic studies of living cells. Membrane continu- the three spinach mtDNA fragments hybridized with approx- ities might facilitate intermolecular recombination; evidence imately equal intensity to MB 11.1 (Fig. 3), the enhancement for both intermolecular and intramolecular recombination of hybridization to spinach Bamr 11.5 appeared to be greater has been accumulated for both mtDNA (1, 29-32) and for the 6.7-kb spinach mtDNA fragment than for the 5.5- and ctDNA (33, 34). Enclosure of the mitochondrion by the chlo- 10.5-kb spinach mtDNA fragments. roplast (29) suggests transformation of the mitochondrion as Thus, it is possible to envision a hierarchical timing of a likely mechanism for ctDNA uptake. DNA sequence transfers, where the ctDNA-homologous On the other hand, exchange of DNA sequences between portion of the 6.7-kb spinach mtDNA fragment was trans- organelles may not require direct physical contact. DNA re- ferred from the chloroplast more recently than the corre- leased into the cytoplasm from broken or lysed chloroplasts sponding parts of the 10.5- and 5.5-kb spinach fragments and may be taken up randomly by the mitochondrion by transfor- where all three spinach homologies represent events that oc- mation. Alternatively, there may exist in the cytoplasm spe- curred in a spinach-specific lineage subsequent to the diver- cific vector molecules capable of transferring sequences be- gence of mung bean and spinach. Alternatively, variable tween organelles. If a vector is facilitating interorganellar rates and patterns of sequence evolution within the mung DNA transfer, it might resemble a transposable element (35) bean and spinach chloroplast genomes could account for the or perhaps a transducing phage, in analogy to tobacco mosa- differential homologies observed. In particular, the portions ic virus, which can produce upon infection a small propor- of MB 11.1 and Bam 11.5 that have homology to the 6.7-kb tion of pseudovirions, which have been shown to contain spinach mtDNA fragment may be more closely related than RNA homologous to both ctDNA and nuclear DNA (36). If are the parts of those ctDNA clones that hybridize to the ctDNA-mtDNA exchange is governed by a vector molecule, 10.5- and 5.5-kb spinach mtDNA Sal I fragments. this might impose a selectivity on which sequences were Among the mtDNAs examined, that of corn has the great- transferred. For instance, a ctDNA sequence flanked by est overall amount of homology to ctDNA (Figs. 1-4). Much strong recombination sites might be transposed more fre- of this strong cross-homology can be explained by invoking quently, as might a sequence with close homology to a viral relatively recent interorganellar DNA transfer. In the case of recognition sequence, such as the encapsidation initiation corn mtDNA hybridization to the ctDNA inverted repeat sequence of tobacco mosaic virus (37). (Figs. 2 and 3), absolute identity of restriction sites has been We feel it unlikely that these integrated ctDNA sequences reported over a 12-kb region of interorganellar homology (6). play any biological role in the mitochondrion. Significantly, In addition, mapping studies indicate that the strong hybrid- the strongest cross-homologies we have observed are to Downloaded by guest on October 1, 2021 2250 Neurobiology: Ferrero et al. Proc. Natl. Acad Sci. USA 81 (1984) A animal model outlined here can be used as a reference model * for GABA receptor studies in man with positron emission 60 computerized tomography. This ex vivo method to measure the binding of [3H]muscimol to high-affinity (nanomolar range) recognition sites in brain labels a single population of GABA recognition sites (Hill slope, 1). This population is formed by GABAA binding sites. In fact, muscimol has a Ee poor affinity for the recognition site of GABA reuptake (17) o 0 40 and for GABAB recognition sites (18). Moreover, studies of the pharmacological specificity of these [3H]muscimol bind- ing sites show displacement by 4,5,6,7-tetrahydroisoxazolo- 30 1U 2U 3u [5,4-c]-pyridin-3-ol and , which are specific GABAA c; Convulsion latency time, min receptor ligands (17, 18), but not by (-), a GABAB receptor ligand (18). We have also found that the high-affini- ty [3H]muscimol binding cannot be measured when the label C Muscimol, umol/kg is injected into mice that received aminooxyacetic acid to increase the brain content of GABA several-fold. We won- dered whether this ex vivo method could be used to study whether diazepam, which increases [3H]muscimol binding

100 when added to brain membranes (3-6), could increase the

S. Bmax of high-affinity [3H]muscimol binding sites in vivo. We '<> 80 found that in mice receiving diazepam, the Bmax of [3H]mus- 0*- cimol binding is doubled. Experiments documenting a rela- tionship between the increase in the number of GABAA rec- 40 4 0 ognition sites and the potentiation of the muscimol-induced 20 ._ inhibition of motor activity showed that these binding sites 4- -k/' that are increased by diazepam are of pharmacological sig- 0.1 0.5 1 5 10 20 nificance. Muscimol, /umol/kg Because several receptor ligands can be metabolized in vivo in compounds with varying degrees of affinity for spe- FIG. 6. (A) Correlation between the in vivo occupancy of GABA recognition sites and the activity of muscimol. Con- cific receptors, it is important that a ligand selected for ex vulsions were induced by subcutaneous administration of isoniazid vivo receptor studies maintains a constant affinity for its re- (2.5 mmol/kg). Muscimol was injected i.v. 20 min after isoniazid. ceptor while present in tissues. In this respect, [3H]musci- Convulsion latency time is the interval in minutes between the injec- mol appears to be an almost ideal ligand for ex vivo experi- tion of isoniazid and the occurrence of tonic-clonic convulsions. ments because its occurs preferentially at the pe- Each point is the mean ± SEM of 8 determinations. *P < 0.05 when riphery and by the mechanism of transamination (13, 21). treated groups are compared with controls (Student's t test). (Inset) The transaminated metabolites are unlikely to enter the Relationship between GABA-receptor occupancy in cerebellum brain, and even if they enter, because of the loss of the ami- (data from Fig. 2) and the prolongation of convulsion latency time no group (13, 21) they will not bind to the recognition site of induced by muscimol. Correlation coefficient (r) = 0.99. (B) Diaze- GABAA receptors. Moreover, the pam enhances muscimol-elicited inhibition of locomotor activity. present experiments show Locomotor activity was recorded 10 min after iv. muscimol injec- that the amount of [3H]muscimol that enters the brain re- mains tion. Diazepam (A) (1.5 Amol/kg, i.p.) or vehicle (A) (control) was practically unmetabolized, perhaps because [3H]mus- injected 5 min before muscimol. Each point (n = 6) is the mean cimol is not taken up by neurons or glial cells. SEM of 10 min of cumulative motility counts. The regional distribution of the number of [3H]muscimol binding sites detected in the ex vivo binding experiments par- allels the regional distribution of the high-affinity GABA rec- but unlike benzodiazepines decreases GABAergic responses ognition sites measured in in vitro experiments (22); howev- (8, 9), fails to increase [3H]muscimol binding measured in ex er, it is difficult to compare the Bmax values calculated from vivo experiments. ex vivo to those calculated from in vitro experiments because Behavioral Studies. To correlate the extent of the ex vivo the in vitro binding studies are carried out under equilibrium binding of muscimol with the intensity of pharmacological conditions and with washed membranes to remove interfer- responses, muscimol was injected i.v. in mice receiving sa- ing material (22); this is not done in ex vivo experiments. line, isoniazid (2.5 mmol/kg, s.c.), or diazepam (1.5 The presence of a diazepam-sensitive mechanism that al- ,umol/kg, i.p.). In mice given saline, a muscimol dose that losterically controls the number of high-affinity binding sites occupies up to 100% of the binding sites fails to modify the for GABA, which was suggested by in vitro experiments (3, gross behavior of mice. However, this dose of muscimol de- 7-9, 23), is confirmed by these ex vivo measurements of lays the onset of isoniazid-induced convulsions (Fig. 6A), GABA binding sites. Diazepam (1.5-3 ,umol/kg, i.p.) rapidly and the muscimol dose required to occupy 50% of the recep- doubles the number of high-affinity binding sites for tors correlates with the ED50 required to decrease isoniazid- [3H]muscimol and at the same time shifts the slope of the elicited convulsion (Fig. 6A, Inset). To confirm that the in- Hill plot from 1 to 1.66, denoting positive cooperativity at crement in the [3H]muscimol binding induced by diazepam the receptor level. Perhaps diazepam produces an allosteric has a pharmacological significance, we have found that a change of GABA recognition sites by altering the inhibitory dose of diazepam that increases the Bmax of muscimol bind- constraint imposed on GABA receptors by GABA-modulin ing causes a shift to the left of the dose-response curve of (3, 4, 23), which we believe functions as an inhibitory cou- muscimol to elicit motor activity impairment (Fig. 6B). pler between the GABA recognition sites and the Cl- chan- nels (24). DISCUSSION The limited number of high-affinity binding sites for mus- This report shows that in mice receiving [3H~muscimol injec- cimol in the different parts of mouse brain suggests that a tions, a uniform population of high-affinity GABA recogni- very small amount of muscimol administered in vivo (0.05- tion sites can be labeled in a saturable manner. Hence, the 0.5 gmol/kg) should be able to saturate the receptors and to Downloaded by guest on October 1, 2021 Neurobiology: Ferrero et aL Proc. NatL. Acad. Sci. USA 81 (1984) 2251

produce maximal pharmacological effects. Indeed, as shown 12. Chang, R. S. L. & Snyder, S. H. (1978) Eur. J. Pharmacol. 48, in Fig. 6, a good correlation exists in mice between the rela- 213-218. tive potency of muscimol to prolong the latency of isoniazid- 13. Baraldi, M., Grandison, L. & Guidotti, A. (1979) Neurophar- induced seizures and to occupy high-affinity GABA recep- macology 18, 57-62. tors as measured by ex vivo binding experiments with 14. Glowinski, J. & Iversen, L. L. (1966) J. Neurochem. 13, 655- [3H]muscimol. 669. 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, 1. Costa, E. & Guidotti, A. (1979) Annu. Rev. Pharmacol. Toxi- R. J. (1951) J. Biol. Chem. 193, 265-275. col. 19, 531-545. 16. Schmid, R., Hong, J. S., Meek, J., & Costa, E. (1980) Brain 2. Haefely, W., Pieri, L., Poic, P. & Schaffner, R. (1981) in Psy- Res. 200, 355-362. chotropic Agents, eds. Hoffmeister, F. & Stille, G. (Springer, 17. Krogsgaard-Larsen, P., Johnston, G. A. R., Lodge, D. & Cur- Berlin), pp. 13-262. tis, D. R. (1977) Nature (London) 268, 53-55. 3. Guidotti, A., Toffano, G. & Costa, E. (1978) Nature (London) 18. Bowery, N. G., Hill, D. R., Hudson, A. L., Doble, A., Midle- 275, 553-555. miss, D. N., Shaw, J. & Turnbull, M. J. (1980) Nature (Lon- 4. Guidotti, A., Forchetti, C. M., Ebstein, B. & Costa, E. (1982) 92-94. in Pharmacology of Benzodiazepines, eds. Usdin, E., Skol- don) 283, nick, P., Tallman, J. F., Greenblatt, D. & Paul, S. M. (Mac- 19. Baxter, C. F. & Roberts, E. (1961) J. Biol. Chem. 236, 3287- millan, Amsterdam), pp. 529-535. 3294. 5. Skerritt, J. H., Willow, M. & Johnston, G. A. R. (1982) Neur- 20. Hunkeler, W., Mohler, H., Pieri, L., Poic, P., Bonetti, E. P., osci. Lett. 29, 63-66. Cumin, R., Schaffner, R. & Haefely, W. (1981) Nature (Lon- 6. Ito, Y. & Kuriyama, K. (1982) Brain Res. 236, 351-363. don) 290, 514-516. 7. Tallman, J. F., Thomas, J. W. & Gallager, D. W. (1978) Na- 21. Moroni, F., Forchetti, M. C., Krogsgaard-Larsen, P. & Gui- ture (London) 274, 383-385. dotti, A. (1982) J. Pharm. Pharmacol. 34, 676-678. 8. Braestrup, C., Schmiechen, R., Neef, G., Nielsen, M. & Peter- 22. Guidotti, A., Gale, K., Suria, A. & Toffano, G. (1979) Brain sen, E. N. (1982) Science 216, 1241-1243. Res. 172, 566-571. 9. Mohler, H. & Richards, J. C. (1981) Nature (London) 294, 23. Guidotti, A., Konkel, D. R., Ebstein, B., Corda, M. G., Wise, 763-765. B. C., Krutzsch, H., Meek, J. L. & Costa, E. (1982) Proc. 10. Pert, C. B. & Snyder, S. H. (1975) Life Sci. 16, 1623-1634. Natl. Acad. Sci. USA 79, 6084-6088. 11. Laduron, P. M., Jansen, P. F. M. & Leysen, J. L. (1978) Bio- 24. Wise, B. C., Guidotti, A. & Costa, E. (1983) Proc. Nati. Acad. chem. Pharmacol. 27, 317-321. Sci. USA 80, 886-890. Downloaded by guest on October 1, 2021