Proc. Nati Acad. Sci. USA Vol. 80, pp. 2072-2076, April 1983 Neurobiology

f8-Carbolines enhance shock-induced suppression of drinking in rats (/phenylpyrazoloquinolines/punished drinking behavior/proconflict action/anxiety) MARIA G. CORDA*, WILLIAM D. BLAKER*, WALLACE B. MENDELSONt, ALESSANDRO GUIDOTTI*, AND ERMINIO COSTA* *Laboratory of Preclinical Pharmacology and tAdult Psychiatry Branch and Clinical Psychobiology Branch, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Contributed by Erminio Costa, December 27, 1982 ABSTRACT By using Vogel's method to test the tenuate the punishment-lessening effect of benzodiazepines in action of benzodiazepines and reducing the intensity of the cur- rats (3) perhaps may even increase fear of punishment in ex- rent delivered to the drinking tube, it is possible to distinguish perimental animals. The present report shows that (3-carboline- the pharmacological activity of three types of ligands for the 3-carboxylic acid methyl ester (3-CCM), 6,7-dimethoxy4ethyl- recognition site. An anticonflict action typical of ,3-carboline-3-carboxylic acid methyl ester (DMCM), frcarbo- anxiolytic benzodiazepines, a proconflict action typical of many line-3-carboxylic acid ethyl ester methyl amide (FG 7142), and (-carbolines, including FG 7142 (3-carboline-3-carboxylic acid ,B3CCE facilitate the suppression induced by punishment, stud- ethyl ester methyl amide), and an antagonistic action of the pro- ied in a behavioral model test for anxiolytic drugs in rats. This conflict and anticonflict actions typical of RO 15-1788 (ethyl-8- fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a]-11,4]-ben- effect of 3-carbolines is blocked by ligands of benzodiazepine zodiazepine-3-carboxylate) and CGS 8216 (2-phenylpyrazolo[4,3- recognition sites that are devoid of intrinsic activity such as RO cjquinolin-3-(5H)-one). Pentylenetetrazole, which causes convul- 15-1788 and CGS 8216 and by ligands with anxiolytic activity. sions by interacting with a subunit of the y-aminobutyric acid receptor that is different from the benzodiazepine recognition MATERIAL AND METHODS site, also induces a proconflict action that is antagonized by anx- Animals. Male Sprague-Dawley rats weighing 120-150 g iolytic benzodiazepines but not by RO 15-1788. were housed in our facilities at 240C with light-dark cycles of 12 hr (light on: 0700-1900) and they were fed Purina Lab Chow A number of ligands that bind with high affinity to the benzo- and water ad lib for at least 3 days before preparation for ex- diazepine recognition site differ from the anxiolytic benzodi- periments. Groups of 16-20 rats to be prepared for the op- azepines pharmacologically (1-10). When a ligand for the ben- erant behavioral tests were deprived of water for 72 hr prior zodiazepine recognition site differs from anxiolytic benzo- to the conflict session. diazepines pharmacologically, it also differs in the way it inter- Conflict-Punishment Procedure. Apparatus. The rat to be acts with y-aminobutyric acid (GABA) receptors biochemically tested was placed in a clear Plexiglas box 28 x 20 x 20 cm (7, 11). Anxiolytic benzodiazepines relieve convulsionsdue toan with a stainless steel grid floor. This chamber was enclosed impairment of GABAergic transmission (12), and increase the in a sound-attenuated ventilated box. Water was provided Bm, (maximal binding) of the high-affinity GABA recogni- through an electrically shielded stainless steel drinking tube tion site; in contrast, the affinity of benzodiazepine recognition extending 2 cm into the box, 3 cm above the floor. The un- sites is increased by GABA (13, 14). A second group of ben- shielded tip of the drinking tube and the grid floor were con- zodiazepine recognition site ligands, such as ethyl-8-fluoro-5,6- nected to a constant current shock generator and to a drink- dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a]-[1,4] -benzodiaze- ometer. The drinkometer output and the shock generator were pine-3-carboxylate (RO 15-1788), 2-phenylpyrazolo[4,3-c]quin- connected to timer controlled relays delivering one shock last- olin-3-(5Hf)-one (CGS '216), and (3-PrCC (,-carboline-3-car- ing for 1 sec for every 3 sec of cumulative drinkometer out- boxylic acid propyl ester) fail to relieve anxiety, bind to the ben- put. This 3-sec cumulative drinking period was termed "lick- zodiazepine recognition site in a GABA-independent manner, ing period." To study the anticonflict drug actions the intensity and fail to modulate the Bma,, of GABA binding (6, 7, 11, 14). of the current delivered to the drinking tube was 0.8 mA; to A third group of benzodiazepine recognition site ligands, such study proconflict drug actions the shock duration was kept as the derivatives of 13-carboline-3-carboxylic acid ethyl ester constant but its intensity was lowered to 0.35 mA. When un- (/3-CCE), elicit or facilitate convulsions, have their affinity for punished drinking was measured the shock was omitted. A the benzodiazepine recognition site reduced by GABA (7), and counter recorded the total number of drinking periods. All block the increase of the Bma, of GABA binding induced by anx- behavioral equipment was purchased from Lafayette Instru- iolytic benzodiazepines (14). One wonders whether these dif- ment, Lafayette, IN. ferences can help to predict the action of various benzodiaze- Procedure. All experiments were carried out between 1100 pine recognition site ligands on anxiety-regulating mechanisms and 1800. One hour before the test, each rat was placed in the in humans. The ability-of drugs to increase the number of pun- in a conflict situation Abbreviations: GABA, y-aminobutyric acid; RO 15-1788, ethyl-8-flu- ished responses in rats operating appears oro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol [1,5-a]-[1,4]-benzodiaze- to be related to. an anxiolytic action in humans (15, 16). Those pine-3-carboxylate; CGS 8216, 2-phenylpyrazolo[4,3-c]quinoin-3-(5H)- f3carbolines that trigger panic-anxiety in humans (7) and at- one; 3,-CCE, ,B-carboline-3-carboxylic acid ethyl ester; 3-CCM, a-car- boline-3-carboxylic acid methyl ester; DMCM, 6,7-dimethoxy4-ethyl- The publication costs of this article were defrayed in part by page charge /-carboline-3-carboxylic acid methyl ester; FG 7142, ,B-carboline-3-car- payment. This article must therefore be hereby marked "advertise- boxylic acid ethyl ester methyl amide; PTZ, pentylenetetrazole; EEG, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. electroencephalogram; i.v., intravenous(ly). 2072 Downloaded by guest on September 27, 2021 Neurobiology: Corda et aL Proc. Natl. Acad. Sci. USA 80 (1983) 2073 chamber and the time needed to find the drinking tube was traperitoneally or subcutaneously, respectively. The injection measured; the rat was removed from the chamber immedi- volume for all the drugs was 3 ml/kg of body weight. De- ately after. Those animals taking more than 3 min to find the pending on the specific time latency for the onset of the drug tube were discarded. Each rat was allowed to habituate to the actions, rats were placed in the test cage either before or im- chamber without the drinking tube for 15 min immediately mediately after drug injection. before the test to avoid exploration-induced delay of drinking. Statistics. Significant differences between control and treated The drinking tube was inserted into the cage and the animals groups were determined as P < 0.05 by using the Dunnett were allowed to complete a 3-sec licking period before the multiple comparison test (19). first shock was delivered. A 3-min test period started at the end of the first shock. Measurement of Threshold to Painful Stimuli. Threshold RESULTS for painful stimuli was measured with the tail flick test of When water-deprived animals were placed in the experimen- D'Amour and Smith (17). The tail flick latency was automat- tal chamber, in the absence of shock, they licked without in- ically determined by a timer to within 1/4 sec. terruption for the test period (3 min), totaling approximately Electroencephalogram (EEG) Recording. Male Sprague- 25 licking periods (Fig. 1A). When a 0.8-mA current was cou- Dawley rats (250 g) were implanted with I/s-inch stainless steel pled every 3 sec to the drinking tube it reduced the number screw electrodes resting on the dura, and 0.005-inch stainless of licking periods significantly (Fig. 1). This conflict situation steel wire electrodes for recording the EEG and nuchal elec- is similar to the one described by Vogel et al. (20). tromyogram (EMG), respectively (1 inch = 25.4 mm). Elec- elicited an anticonflict effect as shown by the reinstatement trode placements were for bipolar bifrontal (channel on trac- of licking activity (Fig. 1B). However, no anticonflict effect ings) and frontooccipital recordings. After a 1-week recovery was detected when f3-carbolines or other nonanxiolytic ligands period, EEG and EMG were recorded starting at 0900 on a of benzodiazepine recognition sites were studied (Fig. 1B). Grass model 78 polygraph calibrated to 50 gV/10 mm and a We then wondered whether some of these ligands induce paper speed of 10 mm/sec. State of consciousness was con- "proconflict effects" by enhancing the suppressing action of ventionally determined (18), and samples of tracings were punishment on drinking. To test this possibility we lowered converted into amplitude vs. frequency plots by an automatic the shock intensity to 0.35 mA because we knew that this cur- fast Fourier transformation apparatus (Bruel and Kjaer, Nae- rent intensity (see Fig. 1A) would minimally decrease spon- rum, Denmark). taneous water drinking and hence allow drug-induced facili- Drugs. The following drugs were used: (-CCE, -CCM, tation of drinking suppression to be more easily observed. FG DMCM, and FG 7142 (gifts from C. Braestrup, Ferrosan, Co- 7142, /3-CCM, DMCM, and j-CCE induced a dose-depen- penhagen, Denmark); diazepam and RO 15-1788 (H. Mohler, dent decrease in the number of shocks received by the ani- Hoffmann-La Roche, Basel, Switzerland); CGS 8216 (B. Pe- mals during the 3-min testing (Fig. 2). track, CIBA-Geigy, Ardsley, NY); pentylenetetrazole (PTZ) The suppression of punished drinking by different /3car- and (Sigma). FG 7142, f3-CCM, f3CCE, DMCM, bolines occurs at dose levels that fail to change unpunished diazepam, RO 15-1788, and CGS 8216 were dissolved in 200 drinking (Fig. 2). Of the ,3carbolines tested, the most potent 1.l of dimethyl sulfoxide and 1 drop of Tween 80, brought up was 3-CCM (IC30, 0.1 mg/kg) and the least potent was FG to volume with saline, and injected intravenously (i.v.). PTZ 7142 (IC30, 1.8 mg/kg). The separation of the changes on and strychnine were dissolved in saline and administered in- punished and unpunished licking elicited by P-CCE was less A B 0.8 mA t

c E 20.A1 E 20

1-u0In -om -00. 0 0 0 0.

. 10- -I

-I

I I I II I I 0J 0 0.2 0.4 0.6 0.8 Current intensity (mA) FIG. 1. (A) Relationship between current intensity and number of licking periods in 3 min. Each point represents the mean + SEM of three to six rats per group. (B) Number of licking periods in 3 min with current intensity set at 0.8 mA in rats receiving i.v. various ligands of the ben- zodiazepine recognition site. Diazepam (0.5 mg/kg), FG 7142 (2.5 mg/kg), CGS 8216 (1 mg/kg), and RO 15-1788 (2 mg/kg) were injected 10 min before the test. Each value represents the mean + SEM of five rats per group. *P < 0.05 compared to the rats performing in an unpunished situation. tP < 0.05 compared to the vehicle-treated group. Downloaded by guest on September 27, 2021 20742gProc.Neurobiology: .Corda et aL Natl. Acad. Sci. USA 80 (1983)

30- FG 7142 30 pCCM Unpunished I~~~~~~ 20- 20

* 10- 10

C

._E 0OJ 0- cm 0' 2 4 NI 0 0.1 0.2 307 30- 0 DMCM L. PCCE 0)

cm._ 204- 20

I10- 10-

0- 0- 0.1 0.2 0 0.2 0.4 Drug (mg/kg i.v.)

FIG. 2. Number of licking periods in 3 min under. punished (o) and unpunished (e) conditions in rats receiving one of four (3-carbolines i.v. FG 7142, /3-CCM, DMCM, and P3CCE were injected 15, 10, 10, and 5 min before the test, respectively: Each point represents the mean ± SEM of 5-11 rats per group. *P < 0.05 compared to the respective vehicle-treated group.

pronounced and more variable than that' observed with the terations in the pain threshold. Doses of (3-carbolines 2-3 times other l-carbolines. At the IC30 dose none of the drugs pro- higher than those required to lower the current threshold that duced readily observable alterations in gross behavior or al- suppresses drinking decreased unpunished drinking and caused A B

PRE-INJECTION

Bifrontal EEG

I-- 0L POST-iNJECTION

Bifrontol EEG FREQUENCY (Hz)

50uV L 1 SEC FIG. 3. EEG effect of ,3-CCM (0.15 mg/kg, i.v.). (A) Bifrontal EEG from control (preinjection) and ,3-CCM (10 minpostinjection) treated animals. (B) The identical EEG signals transformed by fast Fourier analysis into an amplitude vs. frequency plot. The data are from representative animals. FG 7142 (10 mg/kg, iv.) injection produced records similar to control. Downloaded by guest on September 27, 2021 Neurobiology: Corda et aL Proc. Natl.Acad. Sci. USA 80 (1983) 2075

Table 1. Antagonism by RO 15-1788, CGS 8216, and diazepam of DISCUSSION the proconflict effect of (3-carbolines It has been reported that the f3-carbolines RO 15-1788 and Licking periods in 3 min CGS 8216 prevent the anticonflict action of benzodiazepines Antagonist Saline FG 7142 (3CCM DMCM (3, 5, 6, 9, 10). Our experiments indicate that RO 15-1788 and CGS 8216 are almost devoid of intrinsic activity because these Saline 22 ± 2.0 10 ± 0.9* 9 ± 2.1* 11 ± 1.5* two ligands fail to elicit a proconflict or anticonflict action in RO 15-1788 21 ± 1.9 21 ± 2.3 21 ± 1.5 24 ± 1.4 the doses studied here. CGS 8216 18 ± 1.4 17 ± 2.7 16 ± 4.4 15 ± 2.5 In contrast, the present experiments show that f3-CCM, Diazepam 26 ± 2.3 21 ± 2.4 27 ± 1.7 21 ± 2.0 DMCM, FG 7142, and PTZ elicit a proconflict action in rats. RO 15-1788 (2 mg/kg), CGS 8216 (1 mg/kg), diazepam (0.5 mg/kg), The action of f3-carbolines is antagonized by RO 15-1788 and FG 7142 (4 mg/kg), A3CCM (0.15 mg/kg), and DMCM (0.2 mg/kg) were CGS 8216; however, both drugs fail to modify the proconflict injected i.v. 10 min before the test. Each value represents the mean ± activity of PTZ. These findings suggest that, on the basis of SEM of 6-10 animals per group. * P < 0.05 compared to saline-treated group. behavioral action in rats, the benzodiazepine recognition site ligands can be divided into three groups: (i) ligands such as sedation. EEG tracings taken at various times after drug in- diazepam that possess anticonflict action and inhibit the pro- jections indicated that FG 7142 produced no evidence of EEG conflict action of /3-carbolines; (ii) ligands such as P-CCM that or motor sequence activity even in doses up to 10 mg/kg i.v. possess proconflict action and inhibit the anticonflict action of ,(3CCM in doses of 0.15 mg/kg produced occasional brief sharp diazepam; (iii) ligands such as RO 15-1788 and CGS 8216 that wave activity in the EEG but not motor effects at low doses possess neither proconflict nor anticonflict ac- (Fig. 3). Substantially higher doses (0.6 mg/kg) were needed tion, but block the anticonflict and proconflict actions of ben- to elicit major sharp wave activity and motor convulsions. zodiazepines and /3-carbolines, respectively. Thus, it appears The proconflict effects of 13-carbolines appear to be specific that specific ligands are encoded with specific information for because diazepam (0.5 mg/kg, i.v.), RO 15-1788 (2 mg/kg, the benzodiazepine recognition site. If the ligands lack this i.v.), and CGS 8216 (1 mg/kg, i.v.) failed to alter the direction specific information, then they function as simple antagonists of pattern of punished licking under conditions of low current like RO 15-1788 and CGS 8216. This view is upheld by the intensity (Table 1). However, these drugs antagonized the observation that the relative potency of the -carbolines tested suppression of punished drinking elicited by FG 7142, DMCM, in the present experiments correlates positively with their rel- or f3-CCM. ative potency in displacing [3H]flunitrazepam and [3H]diaze- In order to study the specificity of the response induced pam from benzodiazepine binding sites in vitro and in vivo by ,3-carbolines, we have tested PTZ because this drug is known (7). The only exception is ,B-CCE, which appears to be more to affect operant conflict behavior in the rat (21) and to cause potent in the in vitro binding studies than in the behavioral convulsions by reducing GABAergic transmission (22) and experiments. This exception is probably due to its extremely strychnine because this drug reduces glycinergic transmission short half-life in plasma (24). Moreover, the proconflict action (23). As shown in Fig. 4, strychnine in subconvulsive doses of ,B-carbolines is due neither to a decrease in pain threshold failed to alter duration and pattern of punished drinking. In nor to EEG sharp wave activity, insofar as proconflict actions contrast, PTZ significantly decreased the current threshold to occurred with FG 7142 and doses of ,B-CCM lower than those suppress drinking. The rats receiving PTZ doses that fail to that induced sharp wave activity. change unpunished drinking took a smaller number of shocks PTZ, which has been widely used to antagonize the anx- when compared to untreated rats. This effect, however, was iolytic action of benzodiazepines (15), has a pharmacological not reversed by doses of RO 15-1788 that change the pro- profile very similar to that of (3carbolines. Although PTZ is conflict affects of ,3-carbolines (Table 1). chemically unrelated to the ,B-carbolines and has an extremely low affinity for benzodiazepine recognition sites (7), it causes convulsions that are antagonized by benzodiazepines and pro- duces anxiety in humans (21, 25). In the present experiments PTZ causes a proconflict action in doses that are 1/3 to 1/4 of 201- I those required to induce convulsions. However, this procon- c) flict action cannot be antagonized by RO 15-1788 in doses that EUt -Q prevent proconflict effects of but it 0 comparable ,B-carbolines, is inhibited when the benzodiazepine recognition sites are oc- 0. 0_ 0) cupied by anxiolytic benzodiazepine. Electrophysiological .r studies indicate that PTZ is a selective antagonist of GABA- C._ OD co 10- - mediated postsynaptic inhibition (22), conceivably through an -i co inhibition of the opening of the GABA-regulated Cl- chan- ur co 0 0 6 r- nels. On the other hand, strychnine, which inhibits glycine- u I- r%. *- N modulated Cl- conductance (23), has no proconflict effect at to Do- N 0 LI doses close to those producing generalized convulsions. This >I I-- C finding indicates that the proconflict actions elicited by PVZ and ,B-carbolines have similar pharmacological profiles, and electrophysiological studies indicate that both drugs down- FIG. 4. Number of licking periods in 3 minwith electric shock at regulate GABA-mediated postsynaptic responses (22, 26); hence 0.35 mA in rats receiving PTZ or strychnine. PTZ (15 mg/kg, intra- the behavioral actions of ,B-carbolines may be mediated by a peritoneally) was injected 15 minbefore the test. Strychnine (0.6 mg/ down-regulation of GABA receptor function. This conclusion kg, subcutaneously) was injected 20 minbefore the test. RO 15-1788 (2 with that mg/kg, i.v.) was injected 5 minafter the PTZ. Each value represents agrees previous findings /3-carbolines block the ben- the mean + SEM of 8-12 rats per group. zodiazepine-induced enhancement of specific GABA binding *P< 0.05 compared to vehicle-treated group. to synaptic membrane preparations, whereas GABA reduces Downloaded by guest on September 27, 2021 2076 Neurobiology: Corda et al. Proc. Natl. Acad. Sci. USA 80 (1983) the binding of &3-carbolines to the benzodiazepine binding sites 6. Hunkeler, W., Mohler, H., Pieri, L., Pole, P., Bonetti, E. P., (7, 14). Cumin, R., Schaffner, R. & Haefely, W. (1981) Nature (London) 290, 514-516. It has been reported that in the behavioral models of conflict 7. Braestrup, C., Schmiechen, R., Neef, G., Nielsen, M. & Peter- punishment proposed by Geller and Seifter (27) or Vogel et sen, E. N. (1982) Science 216, 1241-1243. al. (20) the anticonflict effect of benzodiazepines is poten- 8. Nutt, D. J. & Cowen, P. J. (1982) Nature (London) 295, 436-438. tiated or mimicked by stimulation of GABA receptors with 9. Petersen, E. N., Paschelke, G., Kehr, W., Nielsen, M. & Braes- (28). Conversely, blockade of GABA receptor func- trup, C. (1982) Eur. J. Pharmacol 82, 217-221. tion with , , or PTZ abolished the anti- 10. Yokoyamy, N., Ritter, B. & Neubert, A. D. (1982)J. Med. Chem. 25, 337-339. conflict action of diazepam (16, 29, 30). These results strongly 11. Mohler, H. & Richards, J. G. (1981) Nature (London) 294, 763- suggest that GABA receptors are operative in the regulation 765. of behavioral inhibition induced by punishment and they are 12. Costa, E., Guidotti, A., Mao, C. C. & Suria, A. (1975) Life Sci. consistent with the view that a facilitation of GABAergic 275, 553-555. transmission mediates the anxiolytic action of benzodiazepines 13. Tallman, J. F., Thomas, J. W. & Gallager, D. W. (1978) Nature (London) 274, 384-385. (31). This facilitation occurs at the postsynaptic GABA recep- 14. Guidotti, A., Forchetti, C. W., Ebstein, B. & Costa, E. (1983) in tor complex, which functions as a supramolecular unit com- Pharmacology of Benzodiazepines, ed. Usdin, E. (Macmillan, New posed of a number of subunits, including GABA recognition York), in press. sites, C1F channel, regulatory sites of this channel, benzo- 15. Cook, L. & Sepinwall, J. (1975) in Mechanism of Action of Ben- diazepine recognition sites, and the coupler protein GABA- zodiazepines, eds. Costa, E. & Greengard, P. (Raven, New York), pp.1-28. modulin (32). Presumably a spectrum of behavioral states 16. Stein, L., Wise, C. D. & Belluzzi, J. D. (1975) in Mechanism of ranging from diazepam-induced anticonflict action to the pro- Action of Benzodiazepines, eds. Costa, E. & Greengard, P. (Raven, conflict action elicited by /3-carbolines and PTZ reflects the New York), pp. 28-44. interaction of the various subunits operative in the supramo- 17. D'Amour, F. E. & Smith, D. L. (1941)J. Pharmacol. Exp. 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