Endogenous benzodiazepine modulation of memory processes

Neuroscience & Biobehavioral Reviews. Vol. 14. pp. 419-424. v Pergamon Press plc. t990. Printed in the U.S.A. 0149-7634/90 $3.00 + .00

Endogenous Benzodiazepine Modulation of Memory Processes

I V A N I Z Q U I E R D O , * C L A U D I O C U N H A * A N D J O R G E H. M E D I N A t

*Centro de Memoria, Departamento de Bioquimica, bzstituto de Biociencias U.F.R.G.S. (centro), 90.049 Porto Alegre, RS, Brasil

"~Laboratorio de Neuroreceptores, Instituto de Biologia Cehdar, Facultad de Medicina Universidad de Buenos Aires, Paraguay 2155, 1113 Buenos Aires, Argentina

IZQUIERDO, I., C. CUNHA AND J. H. MEDINA. Endogenous benzodiazepine modulation of memor 3' processes. NEUROSCI BIOBEHAV REV 14(4) 419--424, 1990.--The immediate posttraining administration of the GABA antagonist, bicuculline, or of the Cl-channel blockers, picrotoxin or Ro 5-4864, enhances memory. These drugs are effective when injected into the amygdaloid nucleus. Intraamygdala muscimol has an opposite effect. All this suggests that memory is modulated at the posttraining period by GABA-A receptors. The pre-, but not posttraining systemic administration of benzodiazepines hinders, and that of inverse agonists, or of the benzodiazepine antagonist, flumazenil enhances retention of diverse tasks. Flumazenil, at doses lower than those that cause an enhancement, antagonizes the effect of benzodiazepine agonists and inverse agonists. This suggests that memory is modulated during acquisition by endogenous benzodiazepine receptor ligands: possibly the diazepam that was recently discovered in brain. Pretraining intraamygdala muscimol administration depresses memory, at doses several times higher than those that are effective posttraining. Pretraining Ro 5-4864 has no effect. This suggests that the release of endogenous benzodiazepines during training may modulate a GABA-A receptor complex, possibly in the amygdala, making it more sensitive to muscimol or Ro 5-4864 in the immediate posttraining period.

Endogenous benzodiazepines GABA-A receptor complex Flumazenil Memory modulation Acquisition Consolidation

GABA is a major inhibitory transmitter in the brain, used perhaps by 30% of all central synapses (43). There are three types of GABA receptor, called GABA-A, B and C (4). Activation of the GABA-A receptor by GABA opens a Cl-channel; the entry of C I - causes postsynaptic inhibition. The GABA-A receptor is the most abundant in the brain and is coupled to regulatory proteic subunits, some of which contain benzodiazepine receptors (43). The composition of the GABA-A receptor complex, in terms of the type(s) of regulatory subunits coupled with the GABA receptor itself, may be different in different brain sites, and thus more or less sensitive to benzodiazepine action (25). There are at least two different kinds of benzodiazepine receptor, one central and one peripheral. The central-type receptor, as its name implies, is predominant in the brain, and binds benzodiazepines, like diazepam, clonazepam and flunitrazepam, and 13-carboline 3-carboxylate esters, like the n-ethyl, n-methyl, and n-butyl esters. Through an allosteric effect, benzodiazepines facilitate, and [3-carboline esters inhibit, the binding of GABA to GABA-A receptors; and thereby enhance and depress, respectively, GABA-mediated postsynaptic inhibition (18, 25, 43). Thus, benzodiazepines are agonists, and [3-carboline esters are inverse agonists at benzodiazepine receptors. The psychopharmacological outcome of the binding of the two classes of substances to the regulatory subunits of the GABA receptor complex is, in fact, opposite: benzodiazepines are anxiolytic and anticonvulsant; 13-carboline esters are anxiogenic and proconvulsant or outright convulsant; etc. (18). The effects and the binding to receptors of both benzodiazepines

and 13-carboline esters are competitively antagonized by flumazenil (Ro15-1788) (16, 18, 22).

The effect of benzodiazepines on memory processes has long been known (26,44). They induce anterograde amnesia without depressing performance during the training session; they are usu- ally ineffective upon posttraining administration (12, 15, 21, 26, 36-38, 44), and their effect is not due to the induction of state dependency (12, 20, 44). [For a discussion of the occasional finding of retrograde amnesia or of suggestions of state dependency, see (20,44).] Thus, the effect of benzodiazepines may be defined as an inhibition of storage resulting from an influence during the acquisition process (15, 20, 21, 44).

In recent years, the effect of [3-carboline esters on memory processes has begun to be studied. Their action is opposite of that of the benzodiazepines. They cause anterograde facilitation (17, 21, 22, 46). Retrograde effects or evidence of state dependency have not been reported. As is the case with most, if not all, learning-enhancing drugs [see (19,28)], at least some [3-carboline esters also seem to have an inverted U dose-response curve, with depressant effects seen at higher doses (17).

The recent discovery of endogenous ligands of benzodiazepine receptors in the brain, including diazepam and n-butyl-13-carboline 3-carboxylate (14), and the observation that the pretraining administration of the competitive antagonist, flumazenil, has effects of its own on learning processes (21, 24, 37), has led to impor- tant new clues on the modulation of acquisition and storage processes. Evidence for posttraining effects of drugs that affect GABA

419

420 IZQUIERDO, CUNHA AND MEDINA

transmission directly, bypassing benzodiazepine receptors, has been available for years (8). Posttraining systemic administration of the Cl-channel blocker, picrotoxin (8,11), or systemic (I 1) or intraamygdala administration of the GABA-A receptor antagonist, bicuculline (10) facilitates retention. This is in contrast to the lack of effect of posttraining benzodiazepines, 13-carbolines or flumazenil (12, 15, 20, 21, 36, 44). As will be seen, recent findings suggest that GABAergic systems regulate memory both at the time of acquisition and at the time of consolidation, but only the former is sensitive to modulation by endogenous benzodiazepine receptor agonists.

ENDOGENOUS BENZODIAZEPINE RECEPTOR LIGANDS: N-BUTYL-I3-CARBOLINE CARBOXYLATE

In 1980, Braestrup and his group isolated n-ethyl-13-carboline 3-carboxylate from brain and urine extracts (5). It was immediately obvious that, since the chemical procedure was quite drastic and involved the use of a methanol-HCl mixture at 80°C during 2 hr, the ethyl ester group was introduced during the extraction (4,5). However, it was also clear that the [3-carboline nucleus might originate in the brain tissue (4, 5, 14, 38). Harmane (2-methyl-13-carboline) and tetrahydro-13-carboline have long been known to exist in brain tissue; the 13-carboline structure may be produced by condensation of aldehydes with indolealkylamines and/or tryptophan [see (14)]. So, even if the ethyl ester was an artifact, the possibility existed that other [3-carboline esters could be formed in the brain.

The ethyl-13-carboline ester studied by Braestrup and his colleagues, and a variety of synthetic analogs, including its amide, which became known as FG 1742, a methyl ester (B-CCM), and methyl-6,7-dimethoxy-4-ethyl-13-carboline-3-carboxylate (DMCM) were found to be inverse agonists at benzodiazepine receptors (4,18). They have psychopharmacological effects opposite to those of the benzodiazepines [see (4-6, 38, 39)]: They cause anxiety in humans, a decrease of the time spent by rats in the open arms of an elevated plus maze (which is taken as a measure of increased anxiety in rats), a facilitation of certain types of learning (17, 21,45), and a proconvulsant or outfight convulsant effect [see (4,17)]. Their effects are blocked by the specific competitive antagonist, flumazenil (Ro15-1788), at doses of the latter that induce no behavioral effect on their own (18).

Recently, De Robertis and his co-workers extracted n-butyl- [3-carboline 3-carboxylate (BCCB) from bovine brain (14, 31, 34). Unlike the previously extracted 13-carboline esters (4,5), BCCB appeared to be a real endogenous substance and not an extraction artifact: It was extracted with cold water at neutral pH (14,34). BCCB was purified by several reverse phase HPLC systems, was identified by u.v. absorption, HPLC, and mass spectrometry, and was shown to displace [3H]-flunitrazepam binding with a K d of about 3 nM and high specificity for benzodiazepine receptors (34). Although it is possible that BCCB may be synthetized in the brain by esterification of the [3-carboline structure (see above), evidence for the biosynthesis of this compound is still lacking (4,14).

BCCB was found to be, like the other [3-carboline esters mentioned above, an inverse agonist at central-type benzodiazepine receptors. In mice, BCCB is proconvulsant (i.e., it lowers the threshold for the induction of convulsions by 3-mercaptopro- pionic acid), and it displays anxiogehic activity, as measured both in the elevated plus maze and by the amount of time spent freezing in an open field; this latter effect is antagonized by a very low dose of flumazenil (32).

Brain BCCB levels increase two-fold after exposure of rats to acute swimming stress, and this change is counteracted by the prior injection of diazepam (30). It is not known whether BCCB is

released by stress (the increased levels could be due to retention rather than release), or whether, if that were the case, it is the only benzodiazepine receptor ligand released by stress. Maximum [3H]-flunitrazepam binding is diminished after the exposure of rats to acute swimming stress (29) or to an elevated plus maze (40), which suggests that some endogenous ligand(s) occupy those receptors during stress.

ENDOGENOUS BENZODIAZEPINE RECEPTOR LIGANDS: BENZODIAZEPINES

De Bias and his co-workers obtained N-desmethyldiazepam, a metabolite of diazepam, from aqueous extracts of bovine and rat brain purified by immunoaffinity chromatography using a monoclonal antibody to the benzodiazepine 3-hemisuccinyloxy-clonazepam (13,42). The substance was identified as N-desmethyldiazepam on various grounds: Competition with [3H]-flunitrazepam binding in a manner undistinguishable from N-desmethyldiazepam, reverse-phase HPLC analysis, u.v. spectrophotometry, and mass spectrometry (42). A compound with the same absortium spectrum and HPLC behavior as oxazepam was also isolated (13,42). Benzodiazepine immunoreactivity was also detected in human and rat brain; including human brains kept in paraffin since 1940, sixteen years before the first industrial synthesis of benzodiazepines (42).

These findings were confirmed by two other groups who used chemical extraction procedures different from those used by De Bias: i.e., conventional purification and extraction methods not employing immunoaffinity. Wildmann et al. (47), and De Rob- ertis and co-workers (14,31) extracted benzodiazepines from bovine and rat brain. In addition, Wildmann et al. found diazepam in rat adrenal medulla (47) and in the plasma of several species, including man (46), and Medina et al. (31) found it in cow mil l and in human milk (Medina, unpublished). The De Robertis group identified diazepam from brain extracts by its behavior in several reverse phase HPLC systems, by its u.v. absortion spectrum, and by the fact that it was recognized by the monoclonal antibody employed by De Bias as if it were diazepam. Perhaps most im- portant of all, Medina et al. (31) found that the diazepam isolated from bovine cerebral gray matter was concentrated in synaptic vesicles, and to a lesser extent, in synaptosome cytosol.

Diazepam and desmethyldiazepam have been found in pota- toes, rice, maize, lentils, milk, soybeans and other plants used as food (45,46), and in cow milk (31), so their presence in the brain may derive from an alimentary origin (14, 42, 45). Microorgan- isms, including some that may contaminate food, synthetize benzodiazepines (27). The detection of diazepam-like substances in plasma (48) suggests that the blood could be the vehicle through which food benzodiazepines reach the brain, as it certainly is the vehicle by which the diazepam ingested as drug reaches the brain. There is no evidence that the brain contains the enzymes needed to synthetize benzodiazepines (14). Therefore, the most parsimo- nious explanation for the presence of diazepam in brain would be to consider it as of alimentary origin. However, De Bias and his co-workers have reported the presence of benzodiazepine immunoreactivity in the neuroblastoma/glioma hybrid cell line NG 108-15 grown for 3 months in a serum (and benzodiazepine) free medium (13), which raises the possibility that it may be synthetized by neurons or glial cells.

The possibility that a putative transmitter may be directly im- ported from food to brain in its final form should not be surpris- ing. Indeed, all neurotransmitters do come from food: The brain amines and the peptides derive from aminoacids; ~he choline of acetylcholine comes with lecithin, etc. In some cases (nora- drenaline, the peptides), several enzymatic steps are needed in

MEMORY MODULATION 421

order to transform the compounds from food into real transmitters; in others (serotonin, histamine), just one or two steps are neces- sary. So, diazepam could well be a case in which no steps are needed in order to transform what is eaten into a transmitter substance.

The finding that a molecule very much like diazepam or diazepam itself is found in synaptic vesicles (31) obviously suggests that it could serve as a neurotransmitter or neuromodu- lator. The suggestion is strengthened by the fact that the benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil), has several psychopharmacological effects of its own, generally opposite to those of the benzodiazepines (16). Prior to the discovery of brain benzodiazepines, these effects were attributed to an inverse agonist intrinsic activity. Indeed, at high doses, flumazenil has effects that may be construed upon as being anxiogenic in laboratory animals: It increases the latency to eat in a novel environment (2), and, most importantly, induces a facilitation of some forms of learning (21, 24, 37, 41), like the 13-carbolines do (17, 21, 22, 46). However, flumazenil is also anticonvulsant, increases resistance to extinction, and is generally without effect in the elevated plus maze test and other behavioral procedures used to measure anxiety in rats or mice (16), all of which are effects opposite to those of the inverse agonists.

While the possibility that flumazenil may have an intrinsic activity of its own (16) cannot be fully ruled out in some cases, particularly at very high doses (i.e., 20 mg/kg or more), the evidence suggesting that brain BCCB and diazepam may be real transmitters opens up the possibility that the effects of fiumazenil on its own could result from an antagonism to endogenous BCCB or diazepam (21, 24, 37). File and Pellow (16) and Medina et al. (31) have suggested that a balance between endogenous benzodiazepine agonist and inverse agonist mechanisms may play a role in the regulation of the perception of, or the response to, anxiety or stress .

Recent findings suggest that this may also be the case for the regulation of learning, at least in circumstances of stress or anxiety. The effects of flumazenil on learning stand out because they are seen at much lower doses than those needed to observe any other psychopharmacological effects, but that are, however, sufficient to antagonize effects of injected BCCB or benzodiazepines.

ENDOGENOUS BENZODIAZEPINE RECEPTOR LIGANDS AND THE REGULATION OF LEARNING

Low, nonanxiogenic doses of flumazenil (5.0 mg/kg or less), given prior to training, enhance retention of habituation to a buzzer (21,37), and active (24) and inhibitory avoidance learning (21,37) in rats (Table 1). The effects have been confirmed in mice using, however, much larger doses (41). Pretraining administration of the naturally occurring inverse agonist, BCCB, also enhances retention of habituation and inhibitory avoidance behavior (21), at doses 5 to 20 times lower than those reported to be anxiogenic or proconvulsant (32). The effect of BCCB is antagonized by a low dose of flumazenil (2.0 mg/kg), ineffective on its own (21). This same low dose of flumazenil also antagonizes the pretraining memory depressant effect of diazepam and clonazepam (21) (Table 1). [Indeed, an antagonism of flumazenil to amnestic effects of diazepam was observed for the first time several years ago by O'Boyle et al. (33) in humans.]

Therefore, the facilitation of acquisition seen with flumazenil on its own suggests that learning of these tasks is normally down-regulated by an endogenous mechanism involving benzodiazepine agonists: In addition to flumazenil having an effect on its own, the tasks are exquisitely sensitive to BCCB.

TABLE 1

EFFECT OF FLUMAZENIL (FLU), BCCB AND CLONAZEPAM (CLO) GIVEN 30 MIN PRIOR TO TRAINING ON RETENTION OF STEP-DOWN

INHIBITORY AVOIDANCE IN RATS

Median (Interquartile Treatment Range) Test Minus Training (mg/kg) N Step-Down Latency (sec)

Vehicle 10 20 (10/31) Flumazenil (2.0) 10 37 (17/180) Flumazenil (5.0) 10 134 (49/300)* BCCB (0.2) 10 69 (47/300)* BCCB (0.5) 10 184 (41/300)* Flu (2.0) + BCCB (0.5) 10 17 (11/29)t Clonazepam (0.4) 10 - 3 ( - 6/3)* Clonazepam (1.0) 10 - 1 ( - 8/8)* Flu (2.0) + CIo (I.0) 10 15 (5/30)t

0.3 mA footshock. Training-test interval, 24 hr. The vehicle was a solution of 10% ethanol, 40% propylene glycol, 0.4% Tween 80, adjusted at pH 7.14 with approximately 5% sodium benzoate/sodium acetate buffer. *Significant difference from its vehicle-control group at p<0.02 in a Mann-Whitney U-test (two-tailed); tSignificant difference from group treated with the same drug and dose without flumazenil at p<0.02 level in a Mann-Whiteney U-test (two-tailed)

This is in line with the findings that mild forms of acute stress, such as swimming (29) or an exposure to the elevated plus maze (40) decrease benzodiazepine receptor binding in the brain. The decreased binding suggests that there may be a release of endogenous benzodiazepine receptor ligands in the brain in those circumstances; and behavioral training procedures are often mildly stressful. Indeed, pretraining flumazenil administration seems to affect retention only of mildly stressful behaviors: It facilitates acquisition of inhibitory avoidance or habituation to a buzzer but not of other obviously less stressful tasks, such as habituation to an open field in rats (21,37) or delayed matching-to-sample in monkeys (41).

Experiments in progress in our laboratories suggest that brain diazepam levels, and [3H]-flunitrazepam binding, may change as a result of training in the behaviors that are sensitive to pretraining flumazenil administration. BCCB brain levels actually increase after acute swimming stress in the rat (30).

Immediate posttraining flumazenil, given at the doses that are effective when given pretraining, has no influence on retention of inhibitory avoidance or habituation to a buzzer in rats (37), and is unable to prevent the effect of pretraining BCCB, diazepam or clonazepam (21) (Table 2). Very high doses of flumazenil (40 mg/kg) have been reported to enhance retention of an avoidance task when given posttraining in mice (41); but with such high doses the possibility of side effects of the drug, including intrinsic activities unrelated to benzodiazepine receptors [see (16)], or anxiogenic effects that may simply add to the reinforcement (19).

The brain localization of the endogenous benzodiazepine sensitive GABAergic mechanism that modulates acquisition is currently being studied. Brioni et al. (9) observed that the intraseptal injection of I or 5 nmoles of muscimol, a GABA mimetic, hinders acquisition of a spatial task (a Morris maze). These doses of muscimol were also effective in reducing high affinity choline uptake by septal cell membranes, suggesting a GABA-cholinergic interaction in the acquisition of this behavior. In another study, 0.5 or 1 nmoles of muscimol injected bilaterally into the amygdaloid nucleus interfered with acquisition of shuttle avoidance in rats (Table 3). The data are as yet insufficient to decide which of these

422 IZQUIERDO, C U N H A AND MEDINA

TABLE 2

EFFECT OF BCCB AND CLONAZEPAM {CLO} GIVEN 30 MIN PRIOR TO TRAINING AND FLUMAZENIL GIVEN IMMEDIATELY POSTrRAINING ON

RETENTION OF STEP-DOWN INHIBITORY AVOIDANCE IN RATS

Treatment Median (Interquartile Range) (mg/kg) Test Minus Training Step- Pretraining Posttraining N Down Latency (see)

Vehicle Vehicle 10 24 (13/55) Vehicle Flumazenil (2.0) 10 20 (13/35) BCCB (0.5) Vehicle l0 135 (98/300)* BCCB (0.5) Flumazenil (2.0) l0 128 (35/300)* Clo (1.0) Vehicle l0 2 ( -4 /3 )* CIo (1.07 Flumazenil (2.07 lO - l ( -4 /3 )*

0.3 mA footshock. Training-test interval, 24 hr. The vehicle was as in Table 1. *Significant difference from the vehicle-control group at p<0.02 level in a Mann-Whitney U-test (two-tailed).

structures is primarily involved, or whether both and/or other structures are involved.

POSTTRAINING MEMORY MODULATION BY DRUGS ACTING ON GABA-A RECEPTORS OR ON THE

GABA-STIMULATED CHLORIDE CHANNEL

Long before picrotoxin was shown to be a blocker of the GABA-act ivated Cl-channel, Breen and McGaugh (8) showed that its posttraining systemic administration facilitates retention of maze learning in rats. The effect was confirmed in a variety of tasks, particularly aversive tasks [see (10,11)]. More recently, the effect of picrotoxin was shown to be shared by the GABA-A receptor antagonist, bicuculline, given either systematically (101 or by intraamygdala injection (11). Muscimol, a G A B A - A agonist. had an opposite effect at very low doses (0.001 nmoles injected into each anaygdaloid nucleus in rats) (11).

Ro 5-4864 (4-chlordiazepam) is both an agonist at peripheral- type benzodiazepine receptors, and a picrotoxin-like blocker of the GABA-A related Cl-channel (1); there is evidence that its intrinsic activity for the latter effect could be quite high, so this effect might

TABLE 3

EFFECT OF INTRAAMYGDALA MUSCINIOL ADMINISTRATION ON RETENTION OF SHUTTLE AVOIDANCE BEHAVIOR IN RATS

Dose Mean ± S.E. Test (nmoles/ Minus Training Number of amygdala) N Shuttle Avoidance Responses

0 9 5 .8 - 1.0 0.001 7 6.6 ~ 2.6 0.01 6 6.2 -'- 2.0 0.1 7 5.7 +- 2.1 0.5 6 - 0 . 3 --- 0.7* 1.0 6 I.l ± 0.9*

Twenty 5-sec tone/0.5 mA, 2-sec footshock trials in both training and test session; training-test interval, 24 hr. Drug dissolved in saline to a volume of 0.5 ,u,l per injection.

Differences in training session performance among groups, not significant at a p<0.1 level: F(5,29)= 1.11.

Differences in test minus training session number of responses (reten- ion scores) significant at a p<0.01 level: F(5,29)= 7.26.

*Significant difference from controls in a Duncan multiple range test at a p<0.0l level.

TABLE 4

EFFECT OF PRE- AND POSTTRAINING 4'-CHLORDIAZEPAM (Ro 5-4864) ADMINISTRATION ON RETENTION OF STEP-DOWN INHIBITORY

AVOIDANCE IN RATS

Treatment. Time and Route of Administration N

Median (Interquartile Range) Test Minus Training Step-Down

Latency (sec)

Intraperitoneal. pretraining: Vehicle 14 15 (10/26) Ro 5-4864, 2.5 mg/kg 11 16 (11/27) Ro 5-4864, 6.25 mgP~g I l 15 (6/17)

Intraperitoneal, posttraining: Vehicle I l 20 (10/23) Ro 5-4864, 1.0 mg/kg 9 34 (5/300)* Ro 5-4864, 2.0 mg/kg 11 47 (41/59)+ Ro 5-4864. 5.0 mg/kg 11 300 (72/300)t

Intracerebroventricular, posttraining: Saline 4 19 (63/707 Vehicle 6 33 (27/35) Ro 5-4864, 2.5 ixg/rat 6 300 (300/300)t

0.3 mA footshock. Training-test interval, 24 hr. The vehicle was as in Table 1. *Significant difference from its vehicle-control group at p<0.01 in a Mann-Whitney U-test (two-tailed); tSame, at p<0.20 level.

predominate at low doses of the substance (1,21). Pretraining systemic administration of large doses of Ro 5-4864 (up to 6.25 mg/kg) has no effect on retention of inhibitory avoidance in rats (21) (Table 4). However , the immediate posttraining administration of much smaller doses of this substance (1 to 5 mg/kg, IP: 8 nmoles, ICV) has a memory facilitatory effect similar to that o f picrotoxin (Table 4). Preliminary findings show that the same effect can be obtained with the intraamygdala injection of as little as 16 pmoles on each side (Cunha, unpublished). Clearly, posttraining mechanisms involved in the modulation of memory, probably in the amygdala, are orders of magnitude more sensitive to muscimol or Ro 5-4864 than the mechanisms affected by pretram|ng mjectmons.

In contrast, with the exception of one experiment using a high dose of f lurazepam (23), posttraining benzodiazepine administration has not been reported to alter retention in a wide variety of tasks and species (12, 15, '44). In our hands [see (15)], posttraining diazepam, midazolam, c lonazepam or flunitrazepam administration hinders memory only at doses that cause both analgesia and a severe ataxia lasting up to or beyond the time of testing.

IS THERE A RELATION BETWEEN THE PRETRAINING AND POSTTRAINING EFFECTS ON MEMORY OF DRUGS ACTING

ON THE GABA RECEPTOR COMPLEX'?

The facts that retention can be affected by benzodiazepine receptor ligands given prior to training, and by G A B A receptor and/or Cl-channel ligands given right after training, and that all these effects seem to, or could, be mediated by the amygdala, suggest that there may be a relation between them.

There are several possibilities, all currently under study in this laboratory. One is that a release of endogenous benzodiazepine agonists (perhaps diazepam) during training may change the state of a GAB A-A receptor complex, possibly in the amygdala, and increase its sensitivity to endogenous GABA or injected muscimol or Ro 5-4864. Thus, the much higher sensitivity of memory to posttraining than to pretraining GABA receptor and Cl-channel ligands, and to pre- rather than to posttraining benzodiazepine receptor ligands, would be easily explained. The s a m e neurones

MEMORY MODULATION 423

could regulate both acquisition and postacquisitional processes involved in storage, by changing their state by an allosteric influence of endogenous benzodiazepines released by the training itself. The passage from acquisition to consolidation would thus involve a change of state of the same neuronal group, namely, GABAergic regulatory cells in the amygdala. The GABAergic synapses would in turn modulate the activity of noradrenergic, cholinergic, or other neurons in that nucleus (28) and/or in its projection sites [see (191].

Another possibility is that there are two distinct GABAergic mechanisms in the amygdala, one active during acquisition, highly sensitive to benzodiazepine agonists and inverse agonists, and another one active at the time of consolidation, very sensitive to muscimol but insensitive to benzodiazepine receptor ligands. This possibility is by far less attractive because it would require the postulation of a type of GABA receptor complex different from those known so far (4): namely, one that is very sensitive to muscimol but insensitive to benzodiazepine ligands.

Still another possibility is that the brain localization of the mechanism involved in the regulation of acquisition and that involved in postacquisitional processes is different. Indeed, pretraining muscimol administration is effective when injected either into the amygdala (Table 1) or into the septum (9): and a variety of drugs effective when given in the posttraining period, like naloxone and opioids are also effective when given either into the amygdala or intraseptally (3). There could well be both a GABAer- gic septal and a GABAergic amygdala system involved in memory modulation to different degrees or interacting in different ways at the time of acquisition and during consolidation. The involvement of two different structures, however, does not necessarily support or explain the finding of different pre- and posttraining sensitivity to muscimol, Ro 5-4864 and benzodiazepine receptor ligands.

Preliminary data from this laboratory favor the first possibility, namely, that of an allosteric change of a GABA-A receptor complex in the anaygdala underlying the transition between the involvement of a neuronal system in acquisition and in consolidation. It might be mentioned in passing that this hypothesis raises an interesting point concerning the relation between acquisition and consolidation: The switch from the former to the latter would include or involve the change of state of a neuronal system involved in the modulation of both.

CONCLUSIONS

Diazepam and its metabolites, N-desmethyldiazepam and oxazepam, are found in the brain of several species, and diazepam is concentrated in synaptic vesicles. Diazepam is also found in plants used as food, and the brain does not seem to have enzymes capable of synthetizing these molecules. Diazepam is also found normally in plasma, which seems thus the vehicle by which it is transported from food to brain. Brain benzodiazepines are contained in synaptic vesicles: therefore, they might well be transmitters involved in the physiological regulation of GABA receptor sites. Recent evidence obtained through the use of the benzodiazepine receptor antagonist, flumazenil (Ro 15-17881 suggests the intriguing possibility that the brain benzodiazepines that apparently come from food may physiologically down-regulate of acquisition during stress (or anxiety: or of anxiogenic tasks; or of tasks acquired in circumstances of stress or anxiety).

Posttraining administration of benzodiazepine receptor inverse agonists, and antagonists, even at high doses, generally has no effect on retention. Posttraining intraamygdala injection of the GABA agonist muscimol depresses, and that of the GABA antagonist bicuculline enhances, retention of inhibitory avoidance in rats. In addition, it has been known for years that posttraining systemic injections of the chloride channel blocker, picrotoxin, enhance retention. This suggests a benzodiazepine-insensitive GABAergic modulation of posttraining memory processing, possibly in the amygdaloid nucleus. The amygdala is sensitive to a great variety of agents that modulate memory: Naloxone, cate- cholamines, etc. [see (19,281].

The relation, if any, of the systems in the amygdala that modulate posttraining memory processing with the apparently physiological mechanism involving endogenous benzodiazepine receptor ligands that regulates acquisition is currently under investigation. One intriguing possibility is that the same GABA-A system, in the anaygdala, may change allosterically by the release of endogenous diazepam during training, and thereby change its role from one in the modulation of acquisition to one in the modulation of postacquisitional processes.

ACKNOWLEDGEMENT

Work supported by a research grant from Fundacao de Amparo a Pesquisa do Rio Grande do Sul (FAPERGSI, Brazil. to I.I.

REFERENCES

1. Basile. A. S.; Bolger, G. T.; Lueddens, H. W. M.; Skolnick, P. Electrophysiological actions of Ro5-4864 on cerebellar Purkinje neurons: Evidence for "'peripheral" benzodiazepine-mediated depres- sion. J. Pharmacol. Exp. Ther. 248:463--469; 1989.

2. Bodnoff. S.; Suranyi-Cadotte, B. E.; Quirion, R.; Meaney, M. J. Role of the central benzodiazepine receptor system in behavioral habituation to novelty. Behav. Neurosci. 103:209-212; 1989.

3. Bostock, E.; Gallagher, M.; King, R. A. Effects of opioid microin- jections into the medial septal area on spatial memory in rats. Behav. Neurosci. 103:643~52; 1989.

4. Bowery, N. GABA-B receptors and their significance in mammalian pharmacology. Trends Pharmacol. Sci. 10:401--407; 1989.

5. Braestrup, C. New developments in the search for central benzodiazepine endogenous ligandIsl. Neurochem. Int. 13:21-24: 1988.

6. Braestrup, C.; Nielsen, M.; Olsen, C. E. Urinary and brain [3- carboline-3-carboxylates as potent inhibitors of brain benzodiazepine receptors. Proc. Natl. Acad. Sci. USA 77:2288-2292; 1980.

7. Braestrup, C.; Schmiechen, R.; Neef, G.: Nielsen, M.; Petersen, E. N. Interaction of convulsive ligands with benzodiazepine receptors. Science 216:1241-1243; 1982.

8. Breen, R. A.; McGaugh. J. L. Facilitation of maze learning with posttrial injections of picrotoxin. J. Comp. Physiol. Psychol. 54: 498-501; 1961.

9. Brioni, J. D.: Decker, M. W.; Gamboa, L.; Izquierdo. I.: McGaugh,

J. L. Muscimol injections in the medial septum impair spatial navigation learning. Soc. Neurosci. Abstr. 15:1173; 1989.

10. Brioni. J. D.; McGaugh, J. L. Posnraining administration of GABAer- gic antagonists enhances retention of aversively-motivated tasks. Psychopharmacology (Berlin) 196:505-510; 1988.

I1. Brioni, J. D.: Nagahara, A.; McGaugh, J. L. Involvement of the amygdala GABAergic system in the modulation of memory storage. Brain Res. 47:105-112; 1989.

12. Cahill, L.; Brioni, J. D.; Izquierdo, I. Retrograde memory enhancement by diazepam: Its relation to anterograde amnesia, and some clinical implications. Psychopharnlacology (Berlin) 90:554-556:1986.

13. De Bias, A. L.; Park, D.; Friedrich, P. Endogenous benzodiazepine- like molecules in the human, rat and bovine brain studied with a monoclonal antibody to benzodiazepines. Brain Res. 413:275-284; 1987.

14. De Robertis, E.; Pena, C.; Paladini, A. C.; Medina, J. H. New developments on the search for the endogenous ligand(s) of central benzodiazepine receptors. Neurochem. Int. 13:1-1 I : 1988.

15. Ferreira, M. B. C. Estudo sobre acao de benzodiazepinas e tiopental sobre memoria. Master's dissertation. Institute of Biosciences. Fed- eral University of Rio Grande do Sul. Porto Alegre: U.F.R.G.S. 1989; 144 pages.

16. File, S. E.; Pellow, S. Intrinsic actions of the benzodiazepine receptor antagonist Ro 15-1788. Psychopharmacology (Berlin) 88: I-11 ; 1986.

424 IZQUIERDO, C U N H A AND MEDINA

17. File, S. E.; Pellow, S. Low and high doses of benzodiazepine receptor inverse agonists respectively improve and impair performance in passive avoidance but do not affect habituation. Behav. Brain Res. 30:31-36; 1988.

18. Haefely, W. In: D. G. Grahame-Smith, ed. Psychopharmacology 2, part 1: Preclinical pharmacology. Amsterdam: Elsevier; 1985:92-182.

19. Izquierdo, I. Different forms of posttraining memory processing. Behav. Neural Biol. 51:171-202; 1989.

20. Izquierdo, I.; Ferreira, M. B. C. Diazepam prevents posttraining drug effects related to state dependency, but not posttraining memory facilitation by epinephrine. Behav. Neural Biol. 51:73-78; 1989.

21. lzquierdo, I.; Pereira, M. E.; Medina, J. H. Benzodiazepine ligand influences on acquisition: Suggestion of an endogenous modulatory mechanism mediated by benzodiazepine receptors. Behav. Neural Biol. 54:27--41; 1990.

22. Jensen, L. H.; Stephens, D. N.; Sarter, M.; Petersen, E. N. Bidirectional effects of 13-carbolines and benzodiazepines on cognitive processes. Brain Res. Bull. 19:359-364; 1987.

23. Jensen, R. A.; Martinez, J. L., Jr.; Vasquez, B. J.; McGaugh, J. L. Benzodiazepines alter acquisition and retention of an inhibitory avoidance response in mice. Psychopharmacology (Berlin) 64:125- 126; 1979.

24. Lal, H.; Kumar, B.; Forster, M. J. Enhancement of learning in mice by a benzodiazepine antagonist. FASEB J. 21:2707-2711; 1988.

25. Levitan, E. S.; Schofield, P. R.: Burt, D. R.; Rhee, L. M.; Wisden, W.; Kohler, M.; Fujita, N.; Rodriguez, H. R.; Stephenson, A.; Darlison, M. G.; Barnard, E. A; Seeburg, P. H. Structural and functional basis for GABA-A receptor heterogeneity. Nature 335: 76-79; 1989.

26. Lister, R. G. The amnesic action of benzodiazepines in man. Neurosci. Biobehav. Rev. 9:87-94; 1985.

27. Luckner, M. Secondary metabolism in microorganisms, plants and animals. 2nd ed. Berlin: Springer; 1984:272-276.

28. McGaugh, J. L. Modulation of memory storage processes. In: Solomon, P. R.; Goethals, G. R.; Kelley, C. M.; Stephens, B. R., eds. Perspectives of memory research. New York: Springer; 1988: 33--64.

29. Medina, J. H.; Novas, M. L.; Wolfman, C.; Levi de Stein, M.; De Robertis, E. Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress. Neuroscience 9:331-335; 1983.

30. Medina, J. H.; Pena, C.; Novas, M. L.; Paladini, A. C.; De Robertis, E. Acute stress induces an increase in rat cerebral cortex levels of n-butyl-13-carboline 3-carboxylate, an endogenous benzodiazepine binding inhibitor. Neurochem. Int. 11:255-259; 1987.

31. Medina, J. H.; Pena, C.; Piva, C.; Paladini, A. C.; De Robertis, E. Presence of benzodiazepine-like molecules in mammalian brain and milk. Biochem. Biophys. Res. Commun. 152:534-539; 1988.

32. Novas, M. L.; Wolfman. C.; Medina, J. H.; De Robertis, E. Proconvulsant and "anxiogenic' effects of n-butyl 13 carboline-3- carboxylate, an endogenous benzodiazepine binding inhibitor from brain. Pharmacol. Biochem. Behav. 30:331-336; 1988.

33. O'Boyle, C.; Lambe, R.; Darragh, A.; Taffe, W.; Brick, I.; Kenny, M. RO 15-1788 antagonizes the effect of diazepam in man without

affecting its bioavailability. Br. J. Anaesth. 55:349-356; 1983. 34. Pena, C.; Medina, J. H.; Novas, M. L.; Paladini, A. C.; De Robertis,

E. Isolation and identification in bovine cerebral cortex of n-butyl 13-carboline-3-carboxylate, a potent benzodiazepine binding inhibitor. Proc. Natl. Acad. Sci. USA 83:4952--4956; 1986.

35. Pereira, M. L.; Dalmaz, C.; Rosat, R. M.; Izquierdo, I. Diazepam blocks the interfering effect of posttraining behavioral manipulations on retention of a shuttle avoidance task. Psychopharmacology (Berlin) 94:402--404; 1988.

36. Pereira, M. E.; Rosat, R.; Huang, C. H.; Godoy, M. G. C.; Izquierdo, 1. Inhibition by diazepam of the effect of additional training and of extinction on the retention of shuttle avoidance behavior in rats. Behav. Neurosci. 103:202-205; 1989.

37. Pereira, M. E.; Medina, J. H.; Izquierdo, I. Effect of pre-training flumazenil administration on retention of three different tasks in rats. Braz. J. Med. Biol. Res. 22:1501-1505; 1989.

38. Prado de Carvalho, L.; Grecksch, G.; Chapoutier, G.; Rossier, J. Anxiogenic and non-anxiogenic benzodiazepine antagonists. Nature 301:64-66; 1983.

39. Prado de Carvalho, L.; Venault, P.; Cavalheiro, E.; Kaijima, M.; Valin, A.; Dodd, R. H.; Potier, P.; Chapouthier, G.; Rossier, J. Distinct behavioral and pharmacological effects of two benzodiazepine antagonists: RO 15-1788 and methyl-[3-carboline. Adv. Bio- chem. Psychopharmacol. 38:175-187; 1983.

40. Rago, L.; Kiivet, R.-A.; Harro, J.; Pold, M. Behavioral differences in an elevated plus-maze: correlation between anxiety and decreased number of GABA and benzodiazepine receptors in mouse cerebral cortex. Naunyn Schmiedebergs Arch. Pharmacol. 337:675--678; 1988.

41. Rumennik, L.; Vincent, G. P.; Schwam, E.; Sepinwall, J. Flumazenil (Ro15-1788) improved memory in mice but not monkeys. Soc. Neurosci. Abstr. 15:1172; 1989.

42. Sangameswaran, L.; Fales, H. M.; Friedrich, P.; De Bias, A. L. Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain. Proc. Natl. Acad. Sci. USA 83:9236-9240; 1986.

43. Sieghart, W. Multiplicity of GABAA-benzodiazepine receptors. Trends Pharmacol. Sci. 10:407--.411; 1989.

44. Thiebot, M. H. Some evidence for amnesic-like effects of benzodiazepines in animals. Neurosci. Biobehav. Rev. 9:95-100; 1985.

45. Unseld, E.; Krishna, D. R.; Fischer, C.; Klotz, U. Endogenous benzodiazepines in brain: right or wrong? Trends Neurosci. 11:490; 1988.

46. Venault, P.; Chapoutier, G.; Prado de Carvalho, L.; Simiand, J.; Morre, M.; Dodd, R. H.; Rossier, J. Benzodiazepine impairs and 13-carboline enhances performance in learning and memory tasks. Nature 321:864-866; 1986.

47. Wildmann, J.; Mohler, H.; Vetler, W.; Ranalder, V.; Schmidt, K.; Maurer, R. Diazepam and N-desmethyldiazepam are found in rat brain and adrenal and may be of plant origin. J. Neural Transm. 70:383-388; 1987.

48. Wildmann, J.; Niemann, J.; Matthaei, H. Endogenous benzodiazepine receptor agonist in human and mammalian plasma. J. Neural Transm. 66:151-160; 1986.

Documents

Endogenous benzodiazepine modulation of memory processes