Chronic benzodiazepine antagonist treatment and its withdrawal upregulates components of GABA-benzodiazepine receptor ionophore complex in cerebral cortex of rat

Brain Research, 519 (1990) 6-11 Elsevier

6

BRES 15542

Chronic benzodiazepine antagonist treatment and its withdrawal upregulates components of GABA-benzodiazepine receptor ionophore complex in cerebral cortex of rat Shrinivas K. Kulkarni and Maharaj K. Ticku Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7764 (U.S.A.)

(Accepted 21 November 1989) Key words: ~'-Aminobutyric acid receptor ionophore complex; Benzodiazepine; Chronic Ro 15-1788 treatment; Withdrawal; Upregulation

Effect of chronic administration of benzodiazepine (BZ) receptor antagonist Ro 15-1788 (flumazenii) (4 mg/kg once daily for 14 days) treatment and its withdrawal on locomotor activity, body temperature, and the binding pattern of receptor ligands that bind to GABA-BZ receptor ionophore complex in different regions of the brain of the rat was studied. Ro 15-1788 (× 14 d) increased the specific binding of [3H]ethy•-8-•u•r•-5-6-dihydr•-5-methy•-6-•x•-4H-imidaz•[••5a][••4]benz•diazepine-3-carb•xy•ate ([3H]Ro 15-1788), [3H]ethyl-8-azido-5-6dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate ([3H]Ro 15-4513), [3H]flunitrazepam, and [aSS]t-butylbicyclophosphorothionate ([35S]TBPS) in cerebral cortex, and this increase in binding remained upregulated during the drug withdrawal at 24 h. The binding of [3H]Ro 15-1788 was also found significantly increased in the hippocampus, but not in cerebellum and striatum. The chronic Ro 15-1788 treatment did not alter the specific binding of [3H]GABA. Rosenthal analysis of the saturation isotherms indicated that the observed upregulation in the binding pattern of [3H]Ro 15-1788 and [~H]Ro 15-4513 in the cerebral cortex was due to an increase in the binding capacity (Bmax). The receptor affinity (Ka) was not changed. The withdrawal of Ro 15-1788 following its chronic administration also enhanced locomotor activity. However, no apparent change in body temperature was observed either due to chronic treatment or withdrawal. These data indicate that chronic Ro 15-1788 treatment and its withdrawal may produce an upregulation of subunits which bind the positive (benzodiazepines), negative (inverse agonist), and neutral (antagonist) ligands of benzodiazepine receptor. This may have an implication in drug discontinuation syndrome due to benzodiazepines. INTRODUCTION Benzodiazepine (BZ) binding sites constitute a part of a supramolecular receptor complex which contains binding sites for y-aminobutyric acid ( G A B A ) , picrotoxin, and barbiturates 2"6'26,34'39'46,49. BZs produce their pharmacological actions by binding to their specific recognition sites and through the facilitation of G A B A e r g i c neurotransmission 2'9'19'28'34'46. Chronic administration of BZs is known to produce tolerance and withdrawal syndromes in humans and laboratory animals lm5'17~ 37,44,47. Tolerance to BZs is reported to be due to the reduction in the ability of BZs to potentiate the inhibitory actions of G A B A , both in vivo and in vitro 17'31. Although the exact mechanism of this receptor function downregulation is not well understood, a change in G A B A - B Z complex and/or a decreased effectiveness of G A B A to activate CI- channels is speculated to be responsible for the downregulation of G A B A - B Z receptor interaction in tolerance 2°'25'31"32'38.

Ro 15-1788, an imidazobenzodiazepine, antagonizes both behavioral and biochemical actions of BZs 35'36. In pharmacological doses it is a neutral ligand for B Z receptors 12, but at higher doses it is reported to possess some intrinsic activity 12'13'28'5°. Ro 15-1788 produces typical antagonist-precipitated withdrawal syndrome when given following chronic B Z administration 12. Although these studies indicate that R o 15-1788 primarily acts at the central B Z receptors, some recent studies have demonstrated differences in binding kinetics and in photoaffinity labeling between this compound and B Z agonist in discrete brain regions 48. A recent study has shown that chronic administration of R o 15-1788 leads to the development of tolerance in behavioral paradigms 13. In the present study, therefore, we investigated the effect of chronic administration of Ro 15-1788 and its withdrawal on the binding of B Z antagonist, [3H]Ro 15-1788 and inverse agonist, [3H]Ro 15-4513, and ligands that bind to the picrotoxin ([35S]TBPS) and G A B A ([3H]GABA) receptor sites in discrete regions of the brain of the rat.

Correspondence: M.K. Ticku, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.

0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

MATERIALS AND METHODS

Materials Male Sprague-Dawley rats weighing 250-300 g maintained on 12 h d/l cycle were used in the present study. The animals had free access to food and water, and were housed at 22 °C under a 12 h light/dark cycle. [3H]GABA (78 Ci/mmol), [3H]Ro 15-1788 (77 Ci/mmol), [3H]Ro 15-4513 (20.1 Ci/mmol), and [35S]TBPS (118.8 Ci/mmoi) were purchased from New England Nuclear (Boston, MA), and [3H]flunitrazepam (85 Ci/mmol) from Amersham (Arlington Heights, IL). Ro 15-1788 was a gift from Hoffmann-La Roche (Nutley, NJ), and Ro 15-4513 was kindly provided by Professor W. Haefely (Hoffmann-La Roche, Basel, Switzerland). Ro 15-1788 and Ro 15-4513 were suspended in DMSO. Bicinchoninic acid (BCA) protein assay reagents were purchased from Pierce Chemical (Rockford, IL). All other chemicals were purchased from commercially available sources.

Chronic Ro 15-1788 administration Ro 15-1788 was dissolved in dimethylsulfoxide (DMSO). Rats were chronically treated with a series of daily pulse injections of Ro 15-1788 (4 mg/kg, i.p.) for 14 days in a volume of I ml/kg. The control rats received an equivalent amount of the vehicle for 14 days. Following chronic treatment with Ro 15-1788, rats were sacrificed 1 and 24 h after the last injection of the BZ-antagonist. Regions of the brain (cerebral cortex, cerebellum, hippocampus and striatum) were dissected and pooled, membranes prepared as described below, and frozen in aliquots for binding studies.

Tissue preparation Tissue preparation for various ligand binding was done as described previouslyTM. Briefly, after appropriate drug treatment schedule, the animals were decapitated and their brains removed and dissected into different regions (cortex, cerebellum, hippocampus and striatum) and pooled. Brain regions were homogenized in 20 ml of ice-cold 0.32 M sucrose and centrifuged at 1000 g for 10 rain. The supernatant was centrifuged at 140 000 g for 30 min to obtain the mitochondrial and microsomal fraction. This fraction was resuspended in ice-cold distilled water and dispersed with a Brinkman Polytron. The suspension was centrifuged at 140 000 g for 30 rain, and the pellet was homogenized in ice-cold water and centrifuged as above. The resulting pellet was suspended in 50 mM Tris-HCl buffer at pH 7.4, centrifuged at 140 000 g for 30 min, and stored in different aliquots of-70 °C. On the day of the experiment, the frozen pellets were thawed at room temperature, suspended in 50 mM Tris-HC1 buffer (pH 7.4), and centrifuged at 140 000 g for 30 rain. The resulting pellets were recentrifuged twice with Tris-HCi buffer (pH 7.4) containing 50 mM KCI. The final pellets were resuspended in the same buffer at a protein concentration of 0.5 to 1.0 mg/ml for binding studies.

Benzodiazepine receptor binding Routinely, [3H]Ro 15-1788 (1 nM), [3H]Ro 15-4513 (1 nM) and [3H]flunitrazepam (1 nM) binding were measured by a filtration assay using Brandel cell harvester, as described previously26'29's°. Briefly, aliquots of tissue homogenates were incubated with 1 nM of various receptor ligands in a total volume of 1 ml for 60 rain at 24 °C. Following incubation, triplicate of 250 /al aliquots were filtered on Whatman GF/B filters and rapidly washed thrice with 3.5 ml buffer washes. The filters were dried at room temperature and radioactivity determined in a 2,5-diphenyloxazole/BBS scintillation cocktail. The nonspecific binding f o r [3H]Ro 15-1788 and [3H]flunitrazepam was determined in the presence of 10 /~M clonazepam and subtracted from the total binding to obtain the specific binding. Similarly, the nonspecific binding for [3H]Ro 15-4513 was determined in the presence of 5 ~uM nonradioactive Ro 15-4513. Saturation isotherms for [3H]Ro 15-1788 and [3H]Ro 15-4513 were done using 0.1, 0.25, 0.5, 1, 2, 5, 10 and 20 nM concentrations

of respective ligands. The Brnax and K d values were obtained by linear regression of the saturation isotherms, using Rosenthal analysis 43.

GABA receptor binding [3H]GABA binding was measured by a centrifugation assay, as described previously7. Briefly, the resuspended tissue preparations were incubated with [3H]GABA (4 nM) in the presence and absence of nonradioactive G A B A (100 gM) in a total incubation volume of 1 ml for 10 rain at 4 °C. Following incubation, the vials were centrifuged at 48 000 g for 10 min. The vials were rapidly washed once and the contents were solubilized with 0.3 ml Soluene-350 (Packard). The radioactivity was determined, as described previously7. Nonspecific binding obtained in the presence of GABA (1130/~M) was subtracted from the total binding to obtain the specific binding.

[35S]t-butylbicyclophosphorothionate binding [35S]TBPS binding was measured by filtration assay, as described previously2z'26. Aliquots of tissue homogenates were incubated with 2 nM [35S]TBPS in a total incubation volume of 1 ml for 180 min at 24 °C. Following incubation, triplicate of 250/A aliquots were filtered on Whatman GF/B filters and washed rapidly with 3.5 ml buffer. The filters were dried and radioactivity determined in a 2,5-diphenyloxazole/BBS (Bio-Solv 3; Beckman) scintillation cocktail. The nonspecific binding, determined in the presence of 10/~M picrotoxinin, was subtracted from the total binding to obtain the specific binding.

Protein estimation Tissue protein was estimated by BCA protein assay 42.

Behavioral studies Effect of acute (1 h) and chronic (× 14 d) treatment with Ro 15-1788 and its withdrawal (24 and 72 h) on locomotor activity was studied using activity meter (Stoelting, U.S.A.). Animals were individually placed in the cages and the locomotor activity was recorded for a 10 min period. Animals were tested 1, 24 or 72 h after the last dose of Ro 15-1788. Each group consisted of 4-8 animals. Any change in body temperature due to Ro 15-1788 or vehicle treatment was recorded using rectal digital thermometer by inserting 3 cm of thermester probe into the rectum.

Statistical analysis Binding data are expressed as mean + S.D, Statistical significance was calculated using Student's t-test.

RESULTS

Behavioral studies Acute

(1 h)

or chronic

(4 m g / k g ,

i.p.

×

14 d)

a d m i n i s t r a t i o n of R o 15-1788 did n o t m o d i f y l o c o m o t o r activity in rats. H o w e v e r , w h e n a n i m a l s w e r e t e s t e d 24 o r 72 h after t h e last d o s e of t h e drug, t h e r e was a significant i n c r e a s e in l o c o m o t o r activity. T h e activity was i n c r e a s e d by 2 9 % o v e r t h e c o n t r o l v a l u e s ( T a b l e I). I n c o n t r a s t , R o 15-1788 did n o t cause any c h a n g e s in r e c t a l t e m p e r a t u r e f o l l o w i n g its a c u t e and c h r o n i c t r e a t m e n t , w i t h d r a w a l (Table I).

or during

Binding studies T h e effect o f c h r o n i c t r e a t m e n t w i t h R o 15-1788 and its w i t h d r a w a l o n b i n d i n g of [ 3 H ] R o 15-1788 in discrete r e g i o n s of the b r a i n is s h o w n in T a b l e II. R o 15-1788

TABLE I

1750 ~.

Effect of acute and chronic treatment with Ro 15-1788 (4 mg/kg, i.p.) and its withdrawal-induced changes on motor activity and body temperature of rats

t500

Motor activity was measured for each rat individually for 10 min using activity meter (Stoelting, U. S.A.). Each group consisted of 4-8 animals.

0.00 h (control) 1h 14d 24 h withdrawal 72 h withdrawal

Motor activity (mean + S.D.)

Body temperature (°C)

1035 1168 1101 1299 1334

37.62 37.54 37.25 37.75 37.81

+ + + + +

126 183 96 151" 86*

+ + + + +

~000

t~

~'

0.4 0.33 0.28 0.28 0.4

~,

PHI Ro~S-~88[b],fmol/mgp,o,ein Fig. 1. Typical Rosenthal plots of the binding of [3H]Ro 15-1788 in the cerebral cortex of control ( 0 ) , chronic treated (O) and withdrawn ( A ) group of animals. The K d and Bin, x values obtained by linear regression are summarized in Table IV.

*P < 0.05, as compared to control (0.00 h).

treatment (x 14 d) increased the specific binding of [3H]Ro 15-1788 in cerebral cortex and hippocampus. This increase in binding remained elevated at 24 h (Table II) and 72 h (data not shown) of drug withdrawal. In contrast, the binding of [3H]Ro 15-1788 in cerebellum and striatum was not significantly altered by chronic drug treatment or its withdrawal (Table II). In the case of inverse agonist [3H]Ro 15-4513, an increase in the binding of the ligand was observed only in the cortex, which remained elevated at 24 h withdrawal. The specific binding of [3H]Ro 15-4513 was not changed in other brain areas, i.e. cerebellum, hippocampus or striatum (Table III). Rosenthal analysis 43 of the data indicated that the increase in [3H]Ro 15-1788 binding in chronically treated or 24 h withdrawal cortex was due to an increase in the number of binding sites (Bmax) (Fig. 1). There was a 32% and 26% increase in Bmax following chronic Ro 15-1788 treatment or its withdrawal, respectively (Table IV). Similarly, the increase in inverse agonist [3H]Ro 15-4513 binding in chronically treated and 24 h withdrawal cortex

T A B L E II

Effect of chronic treatment with Ro 15-1788 (4 mg/kg, i.p.) for 14 days and its withdrawal on binding characteristics of [3H]Ro 15-1788 in different brain regions of rat

TABLE III

Effect of chronic treatment with Ro 15-1788 (4 mg/kg, i.p.) for 14 days and its withdrawal on binding characteristics of [3H]Ro 15-4513 in different brain regions of rat The binding data are mean + S.D. of number of experiments indicated in parenthesis, each done in triplicate. Animals were sacrificed 1 h and 24 h after the last dose (14 day) of drug treatment.

Region

Cortex Cerebellum Hippocampus Striatum

[3H]Ro 15-4513 binding (fmol/mg protein) control

14 d

1216+24 (6) 765 + 52 (12) 733 + 179 (4) 389 + 14 (4)

1285 782 729 438

[3H]Ro 15-1788 binding (fmol/mg protein) control

Cortex Cerebellum Hippocampus Striatum

1069 851 248 447

+ + + +

14 d 49 (12) 47 (3) 15 (3) 38 (6)

*P < 0.05, as compared to control.

1512 886 320 448

24 h withdrawal + + + +

318 (6)* 46 (3) 18 (6)* 104 (4)

1545 + 819 + 329 + 437 +

167 (6)* 24 (3) 33 (9)* 82 (8)

+59(5)* + 24 (4) + 51 (6) +__11 (3)

1579+52(6)* 819 + 52 (4) 721 _+ 53 (3) 426 + 31 (6)

*P < 0.05, as compared to control. TABLE IV

Effect of chronic treatment with Ro 15-1788 (4 mg/kg, i.p.) for 14 days and its 24 h withdrawal (W/D) on binding characteristics of [3H]Ro 15-1788 and [SH] Ro 15-4513 in cerebral cortex of rat Rats were chronically treated with Ro 15-1788, as described in Materials and Methods. [3H]Ro 15-1788 (0.1-20 nM) and [3H]Ro 15-4513 (0.1-20 nM) binding was determined at 23 °C, as described in Materials and Methods. Saturation isotherms were analyzed by Rosenthal method to determine the K d and Bmax values. Values represent the mean + S.D. of at least 3 experiments.

Treatment

The binding data are mean + S.D. of number of experiments indicated in parenthesis, each done in triplicate. Animals were sacrificed 1 and 24 h after the last dose (14 day) of drug treatment.

Region

24 h withdrawal

[3H]Ro 15-1788 Control Ro 15-1788 (× 14 d) Ro 15-1788 (24 h W/D) [3H]Ro 15-4513 Control Ro 15-1788 (× 14 d) Ro 15-1788 (24 h W/D)

Specific binding Bmo~ (fmol/

K~

mg protein)

(nM)

2951 + 64 3896 + 85* 3728 + 125"

2.78 + 0.40 3.78 --_0.46 2.24 + 0.26

4180 + 230 5050 + 578* 4739 + 361"

4.08 + 0.46 5.50 + 1.27 5.06 + 0.70

*P < 0.05, as compared to respective controls.

TABLE V Effect of chronic treatment with Ro 15-1788 (4 mg/kg, i.p.) for 14 days and its withdrawal on binding o f f H]GABA, [3Hjflunitrazepam, and [35Sjt.butylbicyclophosphorothionate (TBPS) in cerebral cortex of rat brain Specific binding of [3H]GABA (4 nM), [3H]flunitrazepam (1 nM), and [35S]TBPS (2 nM) was measured, as described in Materials and

Methods. The binding data are mean + S.D. of number of experiments indicated in parenthesis, each done in triplicate. Animals were sacrificed 1 h and 24 h after the last dose (14 day) of drug treatment. Ligand

[3HIGABA

Specific binding (fmol/mgprotein) control

14 d

24 h withdrawal

254 + 18 (6)

236 + 23 (3)

231 + 4 (3)

770 + 33 (6)* 124 + 24 (6)*

718 + 33 (6)* 105 + 7 (9)*

[3H]Flunitrazepam 619 + 35 (9) [35S]TBPS 82 + 6 (3) *P < 0.05, as compared to control.

was due to an increase in Bmax value (Table IV). The receptor affinity (Kd) for [3H]Ro 15-1788 or [3H]Ro 15-4513 was not significantly altered following chronic Ro 15-1788 treatment or its withdrawal (Table IV). When the overall function of G A B A - B Z receptor ionophore function was studied in cortex using [3H]GABA, [3H]flunitrazepam and [35S]TBPS binding, the binding of [3H]flunitrazepam and [35S]TBPS was found to be significantly increased following chronic administration of Ro 15-1788 to the rats. The binding of these ligands remained elevated at 24 h withdrawal period. However, [3H]GABA binding was unaffected by either chronic drug treatment or its withdrawal (Table V). DISCUSSION

The pharmacodynamic actions of BZs are mediated by high-affinity receptors present on G A B A A chloride ionophore receptor complex2,w. The anxiolytic, anticonvulsant, muscle relaxant, and sedative actions of BZs have been correlated to their ability to bind to their specific recognition sites and subsequent modulation of GABAAergic neurotransmission 2'34'46. Chronic administration of BZ agonists produces tolerance to their clinical, behavioral and electrophysioiogical actions 17' 18,24. Withdrawal reactions sensitive to precipitation by BZ-antagonist have also been reported both in humans and animals ~2. The exact mechanism of tolerance to BZs is not clearly understood. Some of the recent studies have addressed this problem, and contradictory observations have been reported. An earlier study indicated no changes in receptor density or affinity on chronic administration of lorazepam and diazepam 5. Other recent

studies correlated tolerance due to BZs with a reduction in the ability of these agents to potentiate GABAergic transmission 17. Chronic treatment with BZ ligands is reported to reduce the efficacy of coupling between BZ and G A B A A receptors 2°. Chronic diazepam treatment resulted in a decrease in the ability of G A B A to inhibit BZ inverse agonist binding 2°. Further, downregulation of BZ receptor binding and G A B A A receptor function, as assessed by chloride uptake in cortical synaptoneurosomes, may be associated with behavioral tolerance to B Z s 31.

Ro 15-1788, an imidazobenzodiazepine, is a neutral ligand (antagonist) for BZ receptors at pharmacologically relevant doses. It antagonizes behavioral, biochemical, and electrophysiological actions of B Z s 4'9'21'28'35. RO 15-1788 has also been reported to prevent development of chlordiazepoxide withdrawal in rats 1. However, many recent studies have shown that Ro 15-1788 possessed intrinsic activity in a variety of behavioral, neurological, and electrophysiological parameters 12. It has proconvulsant 1° and anxiogenic property ~4 and at higher doses, it may be a partial agonist at BZ receptors 9'28'5°. Ro 15-1788 can precipitate a drug discontinuation syndrome when given after chronic BZ administration ~z. In the present study, we find that chronic treatment with BZ antagonist produces effects opposite to those reported following chronic BZ agonist administration 31" 32 Unlike downregulation of BZ-binding sites and G A B A A receptor function observed with agonists, Ro 15-1788 treatment increased [3H]Ro 15-1788, [3H]flunitrazepam, [3n]Ro 15-4513 and [35S]TBPS binding in cerebral cortex. This increase in receptor density remained upregulated during the withdrawal period as well. Table IV indicates a trend towards a slight decrease in the K d (2.78 nM to 3.78 nM) value of [3H]Ro 15-1788 following chronic treatment. While this difference does not appear to be significant, the possibility of the presence of residual drug in the membranes of chronically treated animals cannot be ruled out. Besides cerebral cortex, the binding of [3H]Ro 15-1788 was found to be increased in hippocampus also. However, in cerebellum and striatum, no change in specific binding was observed either for the antagonist ligand or for the inverse agonist ligand. These observations draw support from other published work and tend to suggest that there could be a regional enhancement of BZ receptor subtypes due to chronic administration of B Z s 33 or other psychotropic agents like ethanol, or during their withdrawal period29" 30. Like BZ agonist withdrawal hyperactivity, Ro 15-1788 withdrawal produced increased locomotor activity. However, chronic treatment or its withdrawal had no effect on other behavioral parameter like body temperature. Several speculations could be made for the observed

10 increase in the Bm~x due to chronic treatment of Ro 15-1788 or following its withdrawal. The G A B A receptor is believed to consist of at least two subunits (a2, f12). Photoaffinity studies have indicated that a-subunit contains the binding sites for BZs and fl-subunit contains the binding sites for G A B A agonists 38'4°. However, it may be noted that both subunits have also been reported to carry binding sites for both G A B A agonists and BZs s. Furthermore, heterogeneity of a-subunits 16 with regional differences 45 has been demonstrated, and these subunits may be coded by distinct genetic codes 23. It is also assumed that all three B Z ligands: positive, neutral, and negative, bind to the a-subunit of the G A B A - B Z receptor complex. While expression of a- or fl-subunits in Xenopus oocytes has been reported to form G A B A A receptor-gated C1--channels, BZs were not effective in potentiating G A B A responses following expression 3'4°. However, a recent study has demonstrated the presence of another subunit, Yz, which apparently binds BZs, and is also responsible for giving functional B Z responses when coexpressed with a 1- or fll-subunits 41. Chronic B Z receptor antagonist treatment may enhance the number or synthesis of recognition sites for these ligands 27. If synthesis of a- and/or y2-subunits of the macromolecular complex were considered to be enhanced due to chronic antagonist treatment, one would expect enhancement in binding of all 3 ligands that bind to these protein subunits. In fact, in the present study the binding of flunitrazepam, R o 15-1788, and Ro 15-4513 was found REFERENCES 1 Baldwin, H.A. and File, S.E., Flumazenil prevents the development Of chlordiazepoxide withdrawal in rats tested in the social interaction test of anxiety, Psychopharmacology, 97 (1989) 424-426. 2 Biggio, G. and Costa, E. (Eds.), Chloride Channels and Their Modulation by Neurotransmitters and Drugs, Raven Press, New York, 1988. 3 Blair, L.A.C., Levitan, E.S., Marshall, J., Dionne, V.E. and Barnard, E.A., Single subunits of the GABA A receptor form ion channels with properties of the native receptor, Science, 242 (1988) 577-579. 4 Bonetti, E.P., Pieri, L., Cumin, R., Schaffner, R., Pieri, M., Gamzu, E.R., Muller, R.K.M. and Haefely, W., Benzodiazcpine antagonist Ro 15-1788: neurological and behavioral effects, Psychopharmacology, 78 (1982) 8-18. 5 Braestrup, C., Nielsen, M. and Squires, R.E, No changes in rat benzodiazepine receptors after withdrawal from continuous treatment with lorazepam and diazepam, Life Sci., 24 (1979) 347-350. 6 Braestrup, C., Nielsen, M., Honore, T., Jensen, L.H. and Petersen, E.N., Benzodiazepine receptor ligands with positive and negative efficacy, Neuropharmacology, 22 (1983) 14511457. 7 Burch, T.P., Thyagarajan, R. and Ticku, M.K., Group-selective modification of the benzodiazepine-GABA receptor-ionophore complex reveals that low affinity GABA receptors stimulate benzodiazepine binding, Mol. Pharmacol., 23 (1983) 52-59. 8 Bureau, M. and Olsen, R.W., y-Aminobutyric acid/benzodiaze-

increased. However, there appears to be a preferential increase towards neutral and negative ligand recognition sites. A n increase in the binding of [35S]TBPS would also be consistent with the involvement of a-subunits, since picrotoxin antagonism of G A B A responses has been demonstrated following expression of a-subunits 3'4°. A selective increase observed in certain regions of the brain in the present study would be consistent with heterogeneity of a-subunits and different coding mechanisms. Although a recent study reported that G A B A A - g a t e d chloride uptake in cortical synaptosomes was also enhanced 33 following chronic administration of B Z receptor antagonist, in the present study [ 3 H ] G A B A binding remained unaltered. However, it may be pointed out that Miller et al. 33 did not measure G A B A binding and they also reported no change in the IC5o values for muscimol inhibition of [35S]TBPS binding. These investigators concluded that chronic R o 15-1788 administration produced no change in the apparent affinity or in coupling to G A B A 33. In summary, our observations, i.e. upregulation of [3H]Ro 15-1788, [3H]Ro 15-4513, [3H]flunitrazepam, and [35S]TBPS binding during chronic treatment of Ro 15-1788 and its withdrawal, may have implications in the development of drug discontinuation syndrome observed in humans and animals. Acknowledgements. We thank Mrs. Diana Reese for excellent secretarial assistance and Dr. Ashok K. Mehta for helpful comments. This work was supported by NIH Grants NS15339 and NS24339. pine receptor protein carry binding sites for both ligands on both two major peptide subunits, Biochem. Biophys. Res. Commun., 153 (1988) 1006-1011. 9 Chan, C.Y. and Farb, D.H., Modulation of neurotransmitter action: control of the 7-aminobutyric acid response through the benzodiazepine receptor, J. Neurosci., 5 (1985) 2365-2373. 10 Corda, M.G., Costa, E. and Guidotti, A., Specific proconvulsant action of an imidazobenzodiazepine (Ro 15-1788) on isoniazid convulsions, Neuropharmacology, 21 (1982) 91-94. 11 File, S.E., Rapid development of tolerance to the sedative effects of lorazepam and triazolam in rats, Psychopharmacology, 73 (1981) 240-245. 12 File, S.E. and PeUow, S., Intrinsic activity of the benzodiazepine receptor antagonist Ro 15-1788, Psychopharmacology, 88 (1986) 1-11. 13 File, S.E., Dingemanse, J., Friedman, H.L. and Greenblatt, D.J., Chronic treatment with Ro 15-1788 distinguishes between its benzodiazepine antagonist, agonist and inverse agonist properties, Psychopharmacology, 89 (1986) 113-117. 14 File, S.E., Lister, R.G. and Nutt, D.J., The anxiogenic action of benzodiazepine antagonists, Neuropharmacology, 21 (1982) 1033-1037. 15 Frey, H.H., Phiilipin, H.P. and Scheuler, W., Development of tolerance to the anticonvulsant effect of diazepam in dogs, Eur. J. Pharmacol., 104 (1984) 27-38. 16 Fuchs, K., Mohler, H. and Sieghart, W., Various proteins from rat brain, specifically and irreversibly labeled [3H]flunitrazepam, are distinct alpha subunits of the GABA-benzodiazepine receptor complex, Neurosci. Lett., 90 (1988) 314-319. 17 Gallager, D.W., Lakoski, J., Gonsalves, S. and Rauch, S.,

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24 25 26

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30

31

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