Benzodiazepine receptor ligand influences on acquisition: Suggestion of an endogenous modulatory mechanism mediated by benzodiazepine receptors

BEHAVIORAL AND NEURAL BIOLOGY 5 4 , 27--41

(1990)

Benzodiazepine Receptor Ligand Influences on Acquisition: Suggestion of an Endogenous Modulatory Mechanism Mediated by Benzodiazepine Receptors IVAN IZQUIERDO,* MARIA ESTER PEREIRA,* AND JORGE H. MEDINAt 3

*Centro de Memoria, lnstituto de Biociencias, U.F.R.G.S. (Centro), 90049 Porto Alegre, RS, Brazil, and tlnstituto de Biologia Celular, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1113) Buenos Aires, Argentina In rats, pretraining ip administration of the central benzodiazepine receptor antagonist, fiumazenil (5.0 mg/kg), or of the inverse agonist, n-butyl-fl-carboline3-carboxylate (BCCB) (0.2 or 0.5 mg/kg), facilitated retention of a step-down inhibitory avoidance task; the central agonists, clonazepam and diazepam (0.4 or 1.0 mg/kg), had an opposite effect, and the peripheral agonist, 4'-chlordiazepam (1.25 or 6.25 mg/kg), was without effect. Pre- but not post-training flumazenil (2,0 mg/kg) blocked the effect of BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), or diazepam (1.0 mg/kg) given also pretraining. The post-training administration of all of these drugs had no effect on retention of the avoidance task. Flumazenil (5.0 mg/kg) and BCCB (0.5 mg/kg), given before training, enhanced retention test performance of habituation to a buzzer but not of habituation to an open field. In the three tasks studied, none of the drugs used had any appreciable effect on training session parameters. These results suggest that there is an endogenous mechanism mediated by benzodiazepine agonists, sensitive to inverse agonists, that normally down-regulates acquisition of certain behaviors; this mechanism becomes activated only when the tasks involve or occur with a certain degree of stress or anxiety (i.e., inhibitory avoidance or habituation to the buzzer) and not in less stressful or anxiogenic tasks (i.e., habituation to an open field). © 1990AcademicPress, Inc.

In chicks, mice, rats, and humans, pretraining administration of diazepam or other benzodiazepines at subataxic doses impairs retention test performance regardless of the type of task, its novelty, its cognitive and motivational components, whether training performance is affected by the drugs, and whether the retention test is carried out in the presence of the drugs (Cahill, Brioni, & Izquierdo, 1986; Izquierdo & Cardoso Supported by a grant from FINEP, Brazil, to I.I. (No. 4.2.88.0273.00) and by a grant from CONICET, Argentina, to J.H.M. We thank Chen I. Huang for her technical assistance and Drs. Claudia Wolfman and Maria Beatriz C. Ferreira for valuable discussions. Address correspondence to Dr. Ivan Izquierdo, Centro de Memoria, Instituto de Bioquimica, Instituto de Biociencias, U.F.R.G.S. (centro), 90049 Porto Alegre, RS, Brazil. 27 0163-1047/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

28

IZQUIERDO, PEREIRA, AND MEDINA

Ferreira, 1989; Lister, 1985; Pereira, Huang, Godoy, & Izquierdo, 1989; Thiebot, 1985; Venault, Chapoutier, Prado de Carvalho, Simiand, Morre, Dodd, & Rossier, 1986). With one exception (Jensen, Martinez, Vasquez, & McGaugh, 1976), effects of posttraining or pretest (Cahill et al., 1986; Izquierdo & Cardoso Ferreira, 1989; Jensen et al., 1976; Thiebot, 1985; Venault et al., 1986) benzodiazepine administrations have not been observed. These findings suggest that benzodiazepines inhibit acquisition (Izquierdo & Cardoso Ferreira, 1989; Jensen et al., 1976; Pereira et al., 1989; Thiebot, 1985; Venault et al., 1986). Some/~-carboline esters are "benzodiazepine inverse agonists": They bind to benzodiazepine receptors but exert behavioral actions that may be called anxiogenic and promote or induce seizures (Braestrup, Nielsen, & Olsen, 1980; File & Pellow, 1986; Novas, Wolfman, Medina, & De Robertis, 1988; Prado de Carvalho, Grecksch, Chapoutier, & Rossier, 1983; Venault et al., 1986). Several/3-carbolines have been reported to enhance retention performance when given at low, presumably nonanxiogenic doses prior to training, but not posttraining or prior to testing. Methyl-/3-carboline-3-carboxylate (/3-CCM) (Venault et al., 1986) enhances inhibitory avoidance and food ingestion in a novel environment (a measure of habituation) in rats and imprinting in chicks (Venault et al., 1986). Methyl-/3-carboline-3-carboxamide (FG1742) and methyl6,7dimethoxy-4-ethyl-/3-carboline-3-carboxylate (DMCM) enhance inhibitory avoidance in mice (Jensen, Stephens, Sarter, & Peterson, 1987) and rats (File & Pellow, 1988) but not holeboard habituation in rats (File & Pellow, 1988). The fact that FG1742 and DMCM impaired retention at higher, and therefore presumably more anxiogenic, doses (File & Pellow, 1988), and the generality of the effect of/3-CCM across different forms of learning (Venault et al., 1986), argue in favor of an influence of the drugs on acquisition perhaps independent of any direct anxiogenic action. Pretraining administration of FG1742 and DMCM prevents scopolamine induced amnesia in an inhibitory avoidance task in rats (Jensen et al., 1987). Pretraining administration of the benzodiazepine receptor antagonist, flumazenil (Ro15-1788), enhances acquisition and retention test performance of active avoidance and reverses scopolamine-induced amnesia of inhibitory avoidance in mice (Lal, Kumar, & Forster, 1988). The enhancement is seen at doses of flumazenil as low as 2.5 mg/kg (Lal et al., 1988), which are not known to be anxiogenic (File & Pellow, 1986; Prado de Carvalho et al., 1983). Another benzodiazepine receptor antagonist, ZK93426, has been reported to have no effect of its own at a high dose (30.0 mg/kg) on inhibitory avoidance in mice, but to counteract both scopolamine- and electroconvulsive shock-induced amnesia at much lower doses (1.0 to 10.0 mg/kg) (Jensen et al., 1987). Although it is possible that these reputed antagonists may have some

BENZODIAZEP1NERECEPTOR LIGANDS AND MEMORY

29

intrinsic activity at benzodiazepine receptors (Jensen, Petersen, Braestrup, Honore, Kehr, Stephens, Schneider, Seidelmann, & Schmiechen, 1984; File & Pellow, 1986), it is generally accepted that flumazenil at low doses (i.e., 5.0 mg/kg or less) has no intrinsic activity of its own (Lal et al., 1988) but is able nevertheless to antagonize the effect both of agonists, like diazepam, and of inverse agonists, like the fl-carbolines. The effect of flumazenil on active avoidance (Lal et al., 1988) suggests that endogenous benzodiazepine agonists may be involved in the regulation of acquisition. Recently, benzodiazepine-like molecules, including diazepam, desmethyldiazepam (De Blas, Park, & Friedrich, 1987; De Robertis, Pena, Paladini, & Medina, 1988; Medine, Pena, Piva, Paladini, & De Robertis, 1988; Sangameswaran, Fales, Friedrich, & De Bias, 1986), and n-butyl-fl-carboline3-carboxylate (BCCB) (Pena, Medina, Novas, Paladini, & De Robertis, 1986; De Robertis et al., 1988) have been detected in the brain. It is possible that the benzodiazepines present in brain are of alimentary origin, since they are also found in cow milk (Medina et al., 1988) and in a variety of plants that serve as food (Sangameswaran et al., 1986; Unseld, Krishna, Fischer, & Klotz, 1988). Several experiments suggest that BCCB is a naturally occurring compound in mammalian brain (De Robertis et al., 1988; Pena et al., 1986) unlike other fl-carbolines, including methyl esters like those mentioned above, which have also been extracted from brain, but appear instead to be extraction artifacts (De Robertis et al., 1988; Braestrup, 1988; Novas et al., 1988). The present paper examines the effect of the ip administration of subproconvulsant, nonanxiogenic doses of BCCB (Novas et al., 1988), of flumazenil (File & Pellow, 1986), of the peripheral benzodiazepine receptor agonist, 4'-chlordiazepam (Ro5-4864) (Pellow & File, 1984), and of low doses of the benzodiazepines, clonazepam and diazepam, on retention of step-down inhibitory avoidance in rats. In addition, the influence of flumazenil and BCCB on two different forms of habituation (to an open field and to a buzzer) was also studied. MATERIAL AND METHODS

Female Wistar rats (406) from our own breeding stock (age, 3 months; median weight, 150 g) were used: 172 in Experiment 1, 110 in Experiment 2, 54 in Experiment 3, 33 in Experiment 4, and 36 in Experiment 5. In Experiments 1, 2, and 3, the animals were trained in a step-down inhibitory avoidance task (Izquierdo & Cardoso Ferreira, 1989; Pereira et al., 1989) and tested 24 h later. The apparatus was a 50 x 25 x 25cm acrylic box with a frontal glass panel and a floor made of parallel 1mm-caliber bronze bars spaced 0.8 mm apart. A 5-cm-high, 7-cm-wide formica platform was placed on the left extreme of the box. During the training session, animals were placed on the platform facing the rear left

30

IZQUIERDO, PEREIRA, AND MEDINA

comer and their latency to step down placing their four paws on the grid was measured with an automatic device. On stepping down, they received a 0.3-mA, 60-Hz, 2-s scrambled footshock and were withdrawn from the box. The test session was similar, only that no footshock was given. Test minus training step-down latency (ceiling, 300 s) was a measure of retention test performance (Izquierdo & Cardoso Ferreira, 1989; Pereira et al., 1989). Experiment 1 examined the effect of flumazenil (Ro15-1788) (2.0 and 5.0 mg/kg), BCCB (0.2 and 0.5 mg/kg), clonazepam (0.4 and 1.0 mg/kg), diazepam (0.4 and 1.0 mg/kg), 4'-chlordiazepam (Ro5-4864) (2.5 and 6.25 mg/kg), and a vehicle (40% propylene glycol, 10% ethanol, and 5% sodium benzoate/benzoic acid to a pH of 7.4), given ip 30 min prior to training, on retention test performance of the avoidance task. Drugs were dissolved in the vehicle. In the case of Ro5-4864, the same vehicle plus Tween 80 (2-3 drops/ml) was used. In this and all following experiments, injection volume was 1.0 ml/kg in all cases. Experiment 2 studied the influence of post-training flumazenil (2.0 mg/kg, ip) or the vehicle on the effect of BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), and diazepam (1.0 mg/kg), given 30 rain prior to training, on retention of the inhibitory avoidance task. Experiment 3 studied the effect of the immediate post-training ip administration of the vehicle, flumazenil (5.0 mg/kg), BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), and diazepam (1.0 mg/kg) on retention of the inhibitory avoidance task. Experiment 4 studied the effect of the vehicle, flumazenil (5.0 mg/kg), and BCCB (0.5 mg/kg), given 30 rain before training, on habituation to an open field. The animals were exposed twice, 5 min each time, to a 40-cm-wide, 50-cmdeep, 60-cm-high open field whose brown linoleum floor was divided into 12 equal quadrangles by white lines. In both sessions, the animals were placed in the rear left quadrangle and their latency to leave that quadrangle was measured with an automatic time counter. The number of line crossings, rearings, and fecal boluses was counted in both sessions (Netto, Dias, & Izquierdo, 1986). The interval between the first exposure (training) and the second exposure to the open field (test session) was 24 h. Experiment 5 studied the influence of the vehicle, flumazenil (5.0 mg/kg), and BCCB (0.5 mg/kg), given ip 30 min prior to training, on habituation to an acoustic stimulus (Izquierdo & Graudenz, 1980). Two boxes similar to those used in Experiments 1, 2, and 3, with no platform, were used. The boxes were 40 cm apart, and a buzzer was placed midway between the two boxes. The animals were placed on the rear left comer of the boxes and allowed to explore freely for 30 s. Immediately after this, the buzzer (90 dB, 2 s) was activated 20 times at random intervals during the next 2 min. Two similar sessions, a training and a test session,

BENZODIAZEPINE RECEPTOR LIGANDS AND MEMORY

31

were carried out with a 4-h interval between sessions. All animals, in all groups, responded to the first buzzer of the training session with a startle, followed by rearing, followed by orienting movements of the head. Startle responses were seen in fact to all buzzers in the first half of the training session. Statistic analysis of the data of Experiments 1, 2, and 3 was by a Kruskal-Wallis analysis of variance followed by individual Mann-Whitney U tests, two-tailed. Analysis of the data of Experiments 4 and 5 was parametric: one-way ANOVAs followed by Newman-Keuls tests and individual t tests where applicable.

RESULTS

Experiment 1. Effect of the Pretraining Administration of Flumazenil, BCCB, Clonazepam, Diazepam, and 4'-Chlordiazepam on Retention The results of Experiment 1 are shown in Table 1A and B. The Kruskal-Wallis analysis of variance showed a significant groups effect for the data of Table 1A (H(ll) = 31.11, p < .01) but not for those of Table 1B (H(2) = 2.26, p > .2). As shown in Table 1A, flumazenil enhanced retention performance at the dose of 5.0 mg/kg; the lower dose (2.0 mg/kg) was ineffective. BCCB enhanced retention at the two doses tested (0.2 and 0.5 mg/kg); the difference between the effect of the two doses was not significant, and the effect of the higher dose was antagonized by flumazenil (2.0 mg/kg). Clonazepam and diazepam impaired retention at the two dose levels studied (0.4 and 1.0 mg/kg); the effect of the lower dose of clonazepam was more marked than that of the same dose of diazepam; and the effect of the higher dose of both benzodiazepines was antagonized by flumazenil (2.0 mg/kg). As shown in Table 1B, the pretest administration of Ro5-4864 (4'chlordiazepam) (2.5 and 6.25 mg/kg) had no effect on retention of the inhibitory avoidance task. The effect of the vehicle with Tween was not significantly different from that of the vehicle without Tween shown in Table 1 A ( U = 119, p > .2). Differences in training step-down latencies among groups were not significant in this experiment (F(14, 157) = 1.55, p > . 1; overall mean, 10.3, range 1.5 to 68.5 s).

Experiment 2. Influence of Post-training Flumazenil on the Effect of Pretraining BCCB, Clonazepam, and Diazepam on Retention The data of Experiment 2 are shown in Table 2. The Kruskal-Wallis analysis revealed a significant groups effect (H(7) = 25.89, p < .01). This experiment replicated part of the findings of Experiment 1: The pretraining administration of BCCB (0.5 mg/kg) enhanced, and that of

32

IZQUIERDO, PEREIRA, AND MEDINA TABLE 1

Treatment (mg/kg)

Retention score (s)

A Vehicle Flumazenil (2.0) Flumazenil (5.0) BCCB (0.2) BCCB (0.5) BCCB (0.5) + ttumazenil (2.0) Clonazepam (0.4) Clonazepam (1.0) Clonazepam (1.0) + flumazenil (2.0) Diazepam (0.4) Diazepam (1.0) Diazepam (1.0) + flumazenil (2.0) Vehicle-tween Ro5-4864 (2.5) Ro5-4864 (6.25)

20.2 ( 9.9/ 31.3) 40.8 (17.4/180.3) 124.4 ( 46.5/300.0)*** 64.3 ( 23.8/300.0)** 187.9 ( 30.9/300.0)*** 12.5 ( 3.9/ 26.6)t -5.8 (-11.4/ 2.9)*** -0.7 - 8 . 5 / 8.4)*** 12.6 3.1/ 29.6)? 10.2 2.0/ 15.5)*# -0.3 - 5 . 2 / 3.2)*** 18.3 9.6/ 48.7)? 15.2 15.7 14,8

9.5/ 26.1) 11.0/ 26.5) 5.5/ 16.5)

Note. (A) Effect of the vehicle, flumazenit (2.0 and 5.0 mg/kg), BCCB (0.2 and 0.5 mg/kg), BCCB (0.5 mg/kg) plus flumazenil (2.0 mg/kg), clonazepam (0.4 and 1.0 mg/kg), clonazepam (1.0 mg/kg) plus flumazenil (2.0 mg/kg), diazepam (0.4 and 1.0 mg/kg), and diazepam (1.0 mg/kg) plus flumazenil (2.0 mg/kg) given ip 30 min prior to training, on retention test performance in a step-down inhibitory avoidance task. Training-test interval, 24 h. Data are expressed as median (interquartile range) test minus training step-down latency. N = 15 for the vehicle group, N = 11 for all other groups. (B) Same as in A using vehicle plus Tween and Ro5-4864 (2.5 and 6.25 mg/kg). * Significant difference from vehicle group, p < .05, in a two-tailed Mann-Whitney U test. **Same, at p < .02 level; ***same, at p < .002 level.tSignificant difference from group treated with the same dose of clonazepam or diazepam without flumazenil, p < .02 level. #Significant difference from group treated with the higher dose of diazepam and from group treated with the same dose of clonazepam, p < .02. Differences between groups in part B, not significant at a p < .2 level. The difference between the control group of part A and that of part B was also not significant at a p < .2 level. c l o n a z e p a m (1.0 m g / k g ) or d i a z e p a m (1.0 m g / k g ) d e p r e s s e d , r e t e n t i o n test p e r f o r m a n c e . P o s t - t r a i n i n g f l u m a z e n i l (2.0 m g / k g ) h a d n o effect of its o w n , a n d w a s u n a b l e to c o u n t e r a c t the p r e t r a i n i n g effects o f the o t h e r drugs. A s in the p r e c e d i n g e x p e r i m e n t , d i f f e r e n c e s in t r a i n i n g l a t e n c i e s a m o n g g r o u p s w e r e also n o t significant: F(7, 102) = 1.21, p > .1; o v e r a l l m e a n , 12.3, r a n g e 1.1 to 50.9 s).

Experiment 3. Effect of Post-training Flumazenil, BCCB, Clonazepam, and Diazepam on Retention T h e findings o f E x p e r i m e n t 3 are s h o w n i n T a b l e 3. T h e K r u s k a l Wallis a n a l y s i s s h o w e d n o significant g r o u p effects (H(4) = 2.02, p >

BENZODIAZEPINE RECEPTOR LIGANDS AND MEMORY

33

TABLE 2 Treatment (mg/kg) pretraining Vehicle Vehicle BCCB (0,5) BCCB (0.5) Clonazepam (1.0) Clonazepam (1.0) Diazepam (1.0) Diazepam (1.0)

Post-training Vehicle Flumazenil Vehicle Flumazenil Vehicle Flumazenil Vehicle Flumazenil

(2.0) (2.0) (2.0) (2.0)

Retention score (s) 25.6 23.5 114.9 165.3 2.4 -1.2 2.8 5.5

( 12.8/ 41.2) ( 8.8/ 23.9) ( 66.6/300.0)** ( 13.3/300.0)** ( - 5 . 3 / 5.2)** ( - 3 . 9 / 3.9)** ( - 1.7/ 5.3)* ( 0.8/ 11.2)*

Note. Effect of the vehicle, BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), and diazepam (1.0 mg/kg), given ip 30 rain prior to training, and of either the vehicle or flumazenil (2.0 mg/kg) given immediately post-training on retention test performance in a step-down inhibitory avoidance task. Training-test interval, 24 h. Data are expressed as median (interquartile range) test minus training step-down latency. N = 15 for the vehicle-vehicle, BCCB (0.5)-vehicle, and BCCB (0.5)-flumazenil groups; N = 13 for all other groups. * Significant difference from vehicle-vehicle group, p < .02, in a two-tailed MannWhitney U test; **same, p < .002.

.2). Post-training flumazenil (5.0 mg/kg), BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), and diazepam (1.0 mg/kg) had no effect on retention. That is, the same doses of these substances that had been found to be effective in Experiments 1 and/or 2 when given prior to training were without effect when given post-training. As in the preceding experiments, training session latency differences among groups were also not significant: F(4, 49) = 3.33, p > .1 (overall mean, 14.9; range, 1.9 to 78.8 s). Experiment 4. Effect of Pretraining Flumazenil and BCCB on Habituation to an Open Field The results of this experiment are shown in Table 4. No training-test differences were observed in the latency to leave the start quadrangle or in defecation in any of the groups (p > .2 in individual t tests). However, significant training-test differences both in the number of rearings and in the number of crossings were observed in all groups (p < .02 to < .001 in t tests), which indicated that there was retention of habituation of these two behaviors. Neither flumazenil (5.0 mg/kg) nor BCCB (0.5 mg/kg), given 30 min prior to training, affected any of the measures of open field activity in any of the two sessions. Differences among groups in the latency to leave the first quadrant, in defecation, in the number of rearings, or in the number of crossings were not significant either in the training or in the test session (see F values in Table 4). Therefore, neither flumazenil nor BCCB, given prior to training at doses that were effective in other

34

IZQUIERDO, PEREIRA, AND MEDINA TABLE 3

Treatment (mg/kg)

Retention score (s)

Vehicle Flumazenil (5.0) BCCB (0.5) Clonazepam (1.0) Diazepam (1.0)

24.4 19.4 17.4 18.1 19.0

(12.3/ 35.8) (7.0/116.1) ( 7.7/ 38.7) ( 5.4/ 22.7) (15.4/ 27.2)

Note. Effect of the vehicle, ltumazenil (5.0 mg/kg), BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), and diazepam (1.0 mg/kg), given immediately post-training, on retention test performance in a step-down inhibitory avoidance task. Training-test interval, 24 h. Data are expressed as median (interquartile range) test minus training step-down latency. N = 10 for the diazepam group; N = 11 for all other groups. Differences between groups not significant in two-tailed Mann-Whitney U tests at p < .2 level.

tasks (see Experiments 1, 2 and 5), had any influence on training or test session performance in the open field. In the case of BCCB this is in contrast with the effect of much larger doses on similar measures of behavior in a single open field session observed in mice by Novas et al. (1988). Experiment 5. Effect of Pretraining Flumazenil and BCCB on Habituation of a Rearing Response to an Acoustic Stimulus Results of this experiment are shown in Table 5. There was evidence of acquisition of habituation in all treatment groups: The number of rearings decreased significantly from the first to the second half of the training session. There was no difference among treatment groups in this decline; i.e., there was no apparent influence of the treatments on the rate of learning of the task. In addition, there was evidence of retention of habituation in all groups: The total number of rearings decreased significantly from the training to the test session. In contrast to the preceding task, the two drugs, given at the same doses, markedly enhanced retention of habituation of rearing to the repeated presentation of a buzzer. Training-test differences were significant in all groups. However, test session performance was much lower in the two drugtreated groups (see F values and results of the NewmanKeuls analysis in Table 2), which indicates an enhanced retention of habituation to the buzzer. The differences between the two drug groups was not significant at a p < .2 level in the Newman-Keuls test.

DISCUSSION In the avoidance task, pre- but not post-training flumazenil (central benzodiazepine receptor antagonist) enhanced retention test performance. Clonazepam (central agonist) and diazepam (central and periph-

0.412

5.2 +_1.1 4.9 _1.0 3.7 _+1.0

Test

0.213

33.6 ± 2.1 38.2 _+ 1.8 39.1 _+ 2.4

Training

0.320

21.1 _+ 2.3 25.1 ± 1.7 27.3 _+ 4.1

Test

N u m b e r o f rearings

1.22

87.1 4.6 103.6 _+ 7.7 99.2 -+ 7.0 _+

Training

0.225

61.4 _+ 5.6 70.4 _+ 7.8 75.9 _+ 6.7

Test

N u m b e r o f crossings

0.096

1.5 _+0.5 1.7 _+0.6 1.5 _+0.5

Training

0.820

2.3 _+0.8 2.0 _+0.7 1.6 _+0.8

Test

N u m b e r of b o l u s e s

Note. Effect of the vehicle, flumazenil (5.0 m g / k g ) , and B C C B (0.5 m g / k g ) given ip 30 min prior to training on behavior in an open field in a 5-min training s e s s i o n and in a 5-min test session. T r a i n i n g - t e s t interval, 24 h. T h e behaviors m e a s u r e d were latency to leave the start q u a d r a n t , n u m b e r of rearings, n u m b e r o f crossings from one q u a d r a n t to another, and n u m b e r of fecal b o l u s e s p e r session. All data are e x p r e s s e d as m e a n s +- SE. N of each group is in parentheses. One-way A N O V A F values are s h o w n for each column. In all cases, differences a m o n g t r e a t m e n t groups were not significant at a p < .2 level. In all t r e a t m e n t groups, t r a i n i n g - t e s t differences in rearings and crossings were significant at a p < .02 level in a t test. T r a i n i n g - t e s t differences in latency values or in the n u m b e r of fecal boluses were not significant at a p < .2 level.

0.516

5.3 _+1.3 4.5 _+0.6 4.1 _+0.6

Vehicle (11) Flumazenil (12) BCCB (10)

F(2, 30)

Training

Treatment

L a t e n c y to leave first quadrant (s)

TABLE4

O

;~

Z

r~

Z r~

N

Z b~ O

33)

0.16

kO.3

5.1

4.4 kO.4 4.6 kO.5

First half

0.26

2.7 kO.4

kO.5

3.1 kO.3 2.9

Second half

0.08

1.5 20.8 7.8 to.6

kO.7

1.5

Total

5

11.98

f 0.3

1.9**

-c 0.5 1.1* f 0.2

2.9

First half

10.55

k 0.3

0.8*

2 0.4

0.9*

k 0.3

2.3

Second half

Rearings in the test session

2.0*

13.57

f 0.4

2.1*

+ 0.3

+ 0.5

5.2

Total

Note. Effect of the vehicle, flumazenil (5.0 mg/kg), and BCCB (0.5 mg/kg) given ip 30 min prior to training on the performance of rearing responses to a 2-s 90-dB buzzer presented 20 times at random intervals in a 2-min training session and in a 2-min test session. Training-test interval, 4 h. Data are expressed as means & SE. N = 12 per group. In all groups, differences between the first and the second half of the training session were significant at a p < .05 level, and differences between total training and total test session performance were significant at a p < .02 level (vehicle group) or at a p < ,001 level (drug groups), both in individual t tests and in a Newman-Keuls test including all data. One-way ANOVA F values are shown for each column. In the training session, differences among treatment groups were not significant at a p < .2 level. In the test session there was a significant treatments effect for all columns at a p < .Ol level. * Significant difference from vehicle group at p < .Ol level in a Newman-Keuls test; **same, at p < .05 level.

m

BCCB

Flumazenil

Vehicle

Treatment

Rearings in the training session

TABLE

28 >

% 5:

P

E E 0

g c t; c “0

BENZODIAZEPINE RECEPTOR LIGANDS AND MEMORY

37

eral agonist) depressed retention, whereas 4'-chlordiazepam (peripheral agonist) had no effect even at high doses. The inverse agonist, BCCB, enhanced retention at very low doses. Clonazepam, diazepam, and BCCB were effective when given pre but not post-training. Pre- but not posttraining flumazenil antagonized the effect of BCCB, clonazepam, and diazepam. Flumazenil and BCCB also enhanced retention of habituation to a buzzer but not of habituation to an open field. None of the drugs had any discernible effect on training session performance in any of the three tasks. The findings on inhibitory avoidance and on habituation to the buzzer, particularly the facts that flumazenil was effective on its own at a low dose and that the tasks were very sensitive to BCCB, suggest that a mechanism involving endogenous benzodiazepine agonists acting at central receptors normally down-regulates acquisition of these behaviors. Obvious candidates for this role are the benzodiazepines found in brain (De Bias et al., 1987; De Robertis et al., 1988; Sangameswaran et al., 1986). File and Pellow (1986) and De Robertis et al. (1988) have suggested that there are two benzodiazepine receptor-mediated systems in the brain, one activated by benzodiazepine-like compounds and the other by/3-carboline-like compounds. If this were so, in the two tasks in which flumazenil had an effect of its own the activity of the benzodiazepinemediated system would predominate over that of the system mediated by inverse agonists. Flumazenil would be expected to block the effects of both agonists and inverse agonists (and of course did so in the present experiment; see Table 1), so obviously the facilitation observed with flumazenil on its own suggests that it was blocking the effect of endogenous agonists. The findings with flumazenil agree with those of Lal et al. (1988), who observed an enhancement of retention of an active avoidance task in mice with pre- but not post-training flumazenil also at low, presumably purely antagonistic, doses. The findings with BCCB agree with those of others who observed an enhancement of inhibitory avoidance (Venault et al., 1986; Jensen et al., 1987; File & Pellow, 1988) and of some (Venault et al., 1986) but not other (File & Pellow, 1988) forms of habituation using low doses of other/3-carbolines. Why are some forms of habituation sensitive to/3-carbolines and flumazenil while others are not? Novelty was clearly not a factor in the present experiments: The three tasks were novel at the time of training, but flumazenil and BCCB affected only two of them. One possibility is that these drugs, and the endogenous modulatory mechanism that their action suggests, affect only the acquisition of behaviors that involve, take place in situations that involve, or require a good perception of stress or anxiety. There is little doubt that inhibitory avoidance is an aversively motivated behavior, stressful and anxiogenic (Dunn, Elsvin,

38

IZQUIERDO, PEREIRA, AND MEDINA

& Berridge, 1986). It may be reasonably argued that habituation to the buzzer was more anxiogenic than habituation to the open field. The buzzer itself may be viewed as mildly aversive: It was loud, and it did induce a startle response at least in the first few presentations. On the other hand, exploring a novel environment and then habituating to it is not particularly stressful; rats respond with freezing rather than with exploration to places that cause them anxiety or stress (Netto et al., 1986). Being exposed to a startling buzzer in a novel environment can be reasonably supposed to be more anxiogenic than being exposed to a novel environment without the buzzer. By the same token, having to eat in a novel environment (Venault et al., 1986) may be considered to be more anxiogenic than just being there with no need to eat. Biological differences (in drug or lesion effects) among different forms of habituation, some more stressful than others, have been described (see Bignami & Michalek, 1978). Loud noises have been known for years to be rather powerful stressors leading to a variety of neuroendocrinologicl consequences (see Selye & Morava, 1952, 1953, for references), whereas the mere exposure to a novel environment does not alter, for example, brain catecholamine levels or metabolism in the rat (Gold, Roberson, & Delanoy, 1985), although it does so in the mouse (Dunn et al., 1986). A study comparing the effect of different forms of habituation on neurohumoral or hormonal correlates of stress or anxiety in different species might be helpful. There was no indication that either flumazenil or BCCB, at the doses used, were anxiogenic. They did not affect training session performance in any of the three tasks studied, including measures such as latency to leave the first quadrant and defecation in the open field experiment. In fact, both drugs were used at doses much below those reported to have anxiogenic or other behavioral effects in rats or mice (File & Pellow, 1986; Novas et al., 1988), including effects on open field behavior (Novas et al., 1988). However, the possibility exists that these two drugs could have had a latent anxiogenic effect in the present experiments (see File & Pellow, 1988), which heightened the perception of or the reaction to anxiogenic or stressful components of the tasks. BCCB and othe /3carbolines, as well as flumazenil (File & Pellow, 1986), although not convulsant per se, may have a latent effect ("proconvulsant") that facilitates the induction of seizures by other substances (Novas et al., 1988; see De Robertis et al., 1988; File & Pellow, 1988). It is notable that the aversive, stressful, or anxiogenic component played an opposite motivational role in the avoidance task and in the habituation to the buzzer. In the avoidance task this component was the actual reinforcement of the task; in habituation to the buzzer, the task consisted precisely in ceasing to respond to a stimulus that lost its aversive or stressful nature with repetition. A heightened perception or "re-

BENZODIAZEPINE RECEPTOR LIGANDS AND MEMORY

39

alization" of what was really anxiogenic and what was not could conceivably contribute to an improved learning of both tasks, regardless of their cognitive content. Pretraining flumazenil, BCCB (Experiments 1, 2, and 5), clonazepam and diazepam (Experiments 1 and 2) affected retention test performance without altering training session parameters. Despite this, it seems more reasonable to attribute their effect to an influence on acquisition than to one on consolidation, since the drugs were ineffective by post-training injection (Experiment 3; see also Thiebot, 1985). There are indications in the literature that training session performance is not necessarily a good measure of how much memory is recorded in each case. A variety of treatments and procedures are known to either enhance or depress training session performance without altering the effective acquisition of many different types of tasks (see Izquierdo, 1989; Pereira et al., 1989, for references). In the particular case of benzodiazepine agonists, inverse agonists and antagonists, both the present findings and nearly all others in the literature showing that they are ineffective administered posttraining seem to be at odds with the many reports that post-training bicuculline and picrotoxin enhance retention (Brioni & McGaugh, 1988). One possibility that is currently being explored in this laboratory is that there may be separate GABAergic modulation of acquisition and of consolidation; the former being much more benzodiazepine-sensitive than the latter. In conclusion, acquisition (at least of some forms of aversive, stressful, or "anxiogenic" learning) appears to be modulated by endogenous benzodiazepine agonist-mediated mechanisms. Flumazenil and BCCB enhance, and benzodiazepines depress, acquisition; and flumazenil antagonizes the effect both of BCCB and of the benzodiazepines. None of these drugs is effective when given post-training. The relation between these effects and the endogenous mechanisms they suggest, and the perception of and/or the reaction to anxiety, deserve further investigation.

REFERENCES Bignami, G., & Michalek, H. (1978). Cholinergic mechanisms and aversively motivated behaviors. In H. Anisman & G. Bignami(Eds.), Psychopharmacology of aversively motivated behaviors (pp. 173-155). New York: Plenum. Braestrup, C. (1988). New developmentsin the search for central benzodiazepineendogenous ligand(s). Neurochemistry International, 13, 21-24. Braestrup, C., Nielsen, M., & Olsen, C. E. (1980). Urinary and brain /3-carboline-3carboxylates as potent inhibitors of brain benzodiazepine receptors. Proceedings of the National Academy of Sciences, U.S.A., 77, 2288-2292. Brioni, J. D., & McGaugh, J. L. (1988). Posttrainingadministrationof GABAergicantagonists enhances retention of aversively-motivatedtasks. Psychopharmacology, 96, 505-510.

40

IZQUIERDO, PEREIRA, AND MEDINA

Cahill, L., Brioni, J. D., & Izquierdo, I. (1986). Retrograde memory enhancement by diazepam: Its relation to anterograde amnesia, and some clinical.implications. Psychopharmacology, 90, 554-556. De Bias, A. L., Park, D., & Friedrich, P. (1987). Endogenous benzodiazepinelike molecules in the human, rat and bovine brain studied with a monoclonal antibody to benzodiazepines. Brain Research, 413, 275-284. De Robertis, E., Pena, C., Paladini, A. C., & Medina, J. H. (1988). New developments on the search for the endogenous ligand(s) of central benzodiazepine receptors. Neurochemistry International, 13, 1-11. Dunn, A. J., Elsvin, K. L., & Berridge, C. W. (1986). Changes in plasma corticosterone and cerebral biogenic amines and their catabolites during training and testing of mice in passive avoidance behavior. Behavioral & Neural Biology, 46, 410-423. File, S. E., & Pellow, S. (1986). Intrinsic actions of the benzodiazepine receptor antagonist Ro15-1788. Psychopharmacology, 88, 1-11. File, S. E., & Pellow, S. (1988). Low and high doses of benzodiazepine receptor inverse agonists respectively improve and impair performance in passive avoidance but do not affect habituation. Behavioral Brain Research, 30, 31-36. Gold, P. E., Roberson, N. L., & Delanoy, R. L. (1985). Post-training brain catecholamine levels: Lack of response to water motivated training. Behavioral & Neural Biology, 44, 425-433. Izquierdo, I. (1989). Different forms of posttraining memory processing. Behavioral & Neural Biology, 51, 171-202. Izquierdo, I., & Cardoso Ferreira, M. B. (1989). Diazepam prevents posttraining drug effects related to state dependency, but not post-training memory facilitation by epinephrine. Behavioral & Neural Biology, 51, 73-78. Izquierdo, I., & Graudenz, M. (1980). Memory facilitation by naloxone is due to release of dopaminergic and beta-adrenergic systems from tonic inhibition. Psychopharmacology, 67, 265-268. Jensen, L. H., Petersen, E. N., Braestrup, C., Honore, T., Kehr, W., Stephens, D. N., Schneider, H., Seidelmann, D., & Schmiechen, R. (1984). Evaluation of the fl-carboline ZK 93426 as a benzodiazepine receptor antagonist. Psychopharmacology, 83, 249256. Jensen, L. H., Stephens, D. N., Sarter, M., & Petersen, E. N. (1987). Bidirectional effects of/3-carbolines and benzodiazepines on cognitive processes. Brain Research Bulletin, 19, 359-364. Jensen, R~ A., Martinez, Jr., J. L., Vasquez, B. J., & McGaugh, J. L. (1976). Benzodiazepines alter acquisition and retention of an inhibitory avoidance response in mice. Psychopharmacology, 64, 125-126. Lal, H., Kumar, B., & Forster, M. J. (1988). Enhancement of learning in mice by a benzodiazepine antagonist. FASEB Journal, 2, 2707-2711. Lister, R. (1985). The amnesic action of benzodiazepines in man. Neuroscience and Biobehavioral Reviews, 9, 87-93. Medina, J. H., Pena, C., Piva, C., Paladini, A. C., & De Robertis, E. (1988). Presence of benzodiazepine-like molecules in mammalian brain and milk. Biochemical and Biophysical Research Communications, 152, 534-539. Netto, C. A., Dias, R. D., & Izquierdo, I. (1986). Training in an open field: Simultaneous learning of habituation and of a water finding task, and differential effect of posttraining naloxone, beta-endorphin, leuenkephalin, and electroconvulsive shock. Psychoneuroendocrinology, 11, 437-446. Novas, M. L., Wolfman, C., Medina, J. H., & De Robertis, E. (1988). Proconvulsant and "anxiogenic' effects of n-butyl/3-carboline-3-carboxylate, an endogenous benzodiazepine binding inhibitor from brain. Pharmacology, Biochemistry & Behavior, 30, 331336.

BENZODIAZEPINE RECEPTOR LIGANDS AND MEMORY

41

Pellow, S., & File, S. (1984). Behavioral actions of Ro5-4864: A peripheraltype benzodiazepine? Life Sciences, 35, 229-240. Pena, C., Medina, J. H., Novas, M. L., Paladini, A. C., & De Robertis, E. (1986). Isolation and identification in bovine cerebral cortex of n-butyl /3-carboline-3-carboxylate, a potent benzodiazepine binding inhibitor. Proceedings of the National Academy of Sciences, U.S.A., 83, 4952-4956. Pereira, M. E., Rosat, R., Huang, C. H., Godoy, M. C. G., & Izquierdo, I. (1989). Effect of additional training on the retention of shuttle avoidance behavior in rats, and the influence of diazepam thereupon. Behavioral Neuroscience, 103, 103-105. Prado de Carvalho, L., Grecksch, G., Chapoutier, G., & Rossier, J. (1983). Anxiogenic and non-anxiogenic benzodiazepine antagonists. Nature(London), 301, 64-66. Sangameswaran, L., Fales, H. M., Friedrich, P., & De Blas, A. L. (1986). Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain. Proceedings of the National Academy of Sciences, U.S.A., 83, 9236-9240. Selye, H., & Morava, A. (1952). Second annual report on stress. Acta, Montreal. Selye, H., & Morava, A. (1953). Ihird annual report on stress. Acta, Montreal. Thiebot, M. H. (1985). Some evidence for amnesic-like effects of benzodiazepines in animals. Neuroscience and Biobehavioral Reviews, 9, 95-100. Unseld, E., Krishna, D. R., Fischer, C., & Ktotz, U. (1988). Endogenous benzodiazepines in brain: right or wrong? Trends in Neurosciences, 11, 490. Venault, P., Chapoutier, G., Prado de Carvalho, L., Simiand, J., Morre, M., Dodd, R. H., & Rossier, J. (1986). Benzodiazepine impairs and/3-carboline enhances performance in learning and memory tasks. Nature(London), 321, 864-866.