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Journal of Psychopharmacology

2000; 14; 288 J PsychopharmacolS. Pompeia, O.F.A. Bueno, L.M. Lucchesi, G.M. Manzano, J.F.C. Galduroz and S. Tufik

similar potenciesA double-dissociation of behavioural and event-related potential effects of two benzodiazepines with

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Introduction

Long-term memory (Graf and Schacter, 1985) comprises anexplicit, or a ‘consciously’ retrievable memory system, and animplicit memory, retrievable without ‘conscious’ control. Explicitmemory is thought to encompass a semantic, or knowledgememory, as well as an episodic memory, or memory for personalexperiences (Graf and Schacter, 1985). Implicit memory, on theother hand, includes phenomena such as conditioning, proceduralmemory and repetition priming (Schacter, 1987). Episodic memoryis the only long-term memory subtype that is affected in amnesicpatients (Shimamura and Squire, 1984) and is susceptible toanterograde impairment by all benzodiazepines (Curran, 1991;Barbee, 1993). These drugs have therefore been used aspharmacological models of organic amnesic syndromes,constituting potential tools in analysing the neurochemicalmechanisms underlying cognitive processes (Lister and File, 1984;Curran, 1991; Danion et al., 1993; Weingartner et al., 1993).

Among all benzodiazepine compounds, lorazepam alone has

been shown to exhibit an atypical mnemonic effect: it consistentlyimpairs perceptual priming (hereafter referred to only as priming)as measured, mainly, by indirect stem-completion tasks, althoughin many cases this drug leads to similar psychomotor andsubjective effects to other benzodiazepines (Curran andGorenstein, 1993; Legrand et al., 1995; Stewart et al., 1996;Buffett-Jerrott et al., 1998). The reason why lorazepam exhibitsthis atypical effect is unclear. It may relate to specific unknownpharmacological characteristics of this drug, which may lead todistinct effects from those of other benzodiazepines, or beassociated with the nature of the stem-completion task used toassess priming.

In the indirect stem-completion task, subjects are initiallyshown a series of words and are then instructed at test to completethree-letter word-stems with the first word that comes to mind. Ingeneral, half the stems complete words that were previously shown(primed words), while the remaining stems complete words thatwere seen by other subjects (unprimed words) so that base-ratecompletion can be established. Memory is measured by the

Journal of Psychopharmacology 14(3) (2000) 288–298

©2000 British Association for Psychopharmacology (ISSN 0269-8811)

SAGE Publications, London, Thousand Oaks, CA and New Delhi

0269–8811[2000009]14:3; 288-298; 014448

A double-dissociation of behavioural and event-related potential effects

of two benzodiazepines with similar potencies

S. Pompéia1, O. F. A. Bueno1, L. M. Lucchesi1, G. M. Manzano2, J. C. F. Galduróz1 and S. Tufik1

1Departamento de Psicobiologia, UNIFESP, Brazil, 2Setor de Neurofisiologia Clínica, Departamento de Neurologia, UNIFESP, Brazil.

This study was designed to explore the role of benzodiazepine affinity to benzodiazepine binding site on

acute psychomotor, subjective and memory effects, as well as auditory Event Related Potential (ERP)

latencies, in healthy volunteers. Two benzodiazepines with similar affinity to benzodiazepine binding sites,

or potency, were compared: the atypical compound lorazepam (2.0 mg), which has been reported to impair

priming, and a standard benzodiazepine, flunitrazepam (0.6 mg, 0.8 mg, 1.0 mg). The study followed a

placebo-controlled, double-blind, parallel-group design. Sixty subjects completed a test battery before

treatment and at theoretical peak plasma concentration of drugs. Lorazepam and 1.0 mg of flunitrazepam

led to comparable alterations on psychomotor, subjective and auditory episodic memory measures. A

double-dissociation was found for lorazepam and the equipotent dose of flunitrazepam (1.0 mg): lorazepam

was more deleterious than flunitrazepam in time taken to identify fragmented shapes. Lorazepam also

impaired direct and indirect stem-completion in comparison to placebo, but this effect was abolished when

time to identify shapes was used as a covariate. By contrast, 1.0 mg of flunitrazepam prolonged auditory ERP

latencies to a greater extent than lorazepam. High affinity to the benzodiazepine binding sites does not

seem to explain the consistent lorazepam-induced impairment of indirect stem-completion. Differences in

impairment profile between the benzodiazepines employed may relate to the modality (visual or not) of the

tasks used.

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difference in completions between primed and unprimed words.This task, however, is not an ideal measure of priming becausenormal subjects can employ both implicit (priming) and explicit(episodic) strategies while carrying it out (Richardson-Klavehn andBjork, 1988). It is therefore difficult to establish to what extentreductions induced by lorazepam in stem completions with wordsseen before are due to impaired episodic memory, which is affectedby all benzodiazepines, or to implicit memory changes. This iscomplicated by the fact that lorazepam’s memory profile has onlybeen compared with that of other benzodiazepines such asdiazepam (Sellal et al., 1992; Vidailhet et al., 1994; Legrand et al.,1995; Vidailhet et al., 1996) and oxazepam (Curran andGorenstein, 1993; Stewart et al., 1996), drugs that exhibit lowerpotency or affinity to the central benzodiazepine binding sites onGABAA receptor, a pharmacological characteristic that mayaccount for the extent of benzodiazepine mnemonic effects (Curranet al., 1987; Vgontzas et al., 1995; Pompéia et al., 1996a,b).Hence, it would be advisable to compare lorazepam to anotherbenzodiazepine with similar potency in order to determine whetherlorazepam’s atypical effects on indirect stem-completion are due toits high receptor affinity, and therefore pronounced episodicmemory effects, or to specific impairment of priming.

Specific methodologies such as the ‘Retrieval IntentionalityCriterion’ (RIC; Schacter et al., 1989) must be employed in orderto determine the occurrence of contamination of episodic memoryin indirect tasks. This paradigm requires a dissociation betweensubtypes of memory using direct and indirect versions of taskswhen all conditions except task instructions are held constant (e.g.direct and indirect stem-completion). The advantage in using theRIC is that the constant presentation/test conditions enable directcomparison of performance on direct and indirect tasks. Amanipulation known to effect explicit and implicit memorydifferently, such as Levels of Processing (LOP; Flory and Pring,1995), must also be applied at encoding. Because semanticprocessing is thought to facilitate explicit retrieval while leavingimplicit memory unchanged (Bishop and Curran, 1995; Hamannand Squire, 1996), if performance in indirect tests does not benefitfrom semantic encoding, it may be said that explicit memory didnot contaminate results.

Few studies have employed strategies such as the RIC toevaluate benzodiazepine mnemonic effects. In one such study,Bishop and Curran (1995) reported that lorazepam impairedindirect stem-completion when no differences between semanticand perceptual encoding were found, lending support to theassertion that lorazepam does in fact impair implicit memory anddoes not simply diminish contamination of explicit strategies inthis task. Comparisons of lorazepam and another benzodiazepine,oxazepam, have also been made using direct and indirect stem-completion but with no manipulation of LOP (Stewart et al., 1996;Buffett-Jerrott et al., 1998), precluding the possibility of examiningcontamination of explicit memory in indirect stem-completion. Inaddition, the RIC has not been applied to compare lorazepam and abenzodiazepine with similar potency.

Apart from changes in episodic memory, benzodiazepines alsolead to psychomotor (Koelega, 1989; Kunsman et al., 1992) andsubjective alterations including sedation, which is often indexed bypsychomotor impairment and subjective ratings and can contributeto benzodiazepine-induced reduction of memory (Curran, 1991;Barbee, 1993). However, changes in arousal can be dissociatedfrom amnesic effects in various ways such as by covarying

measures of sedation from memory scores (Curran, 1991).Alternative aspects of information processing, which are

relatively inaccessible using self-report or traditional behaviouraltesting, can be evaluated using Event Related Potentials (ERPs).According to Pfefferbaum et al. (1995), the ERPs are generated byslowly changing membrane potentials of dendrites and nerve cellbodies that are time-locked to sensory, motor and cognitive events.These changes in potentials generate extracellular current flow thatis summated in volume-conducted potentials recorded at the scalp.ERPs are usually measured as peaks and troughs, reliably observedpositive or negative deflections related to stimuli (Pfefferbaum etal., 1995).

The effects of various benzodiazepines on ERPs have beeninvestigated and increases in latency and decreases in amplitudehave been reported at various scalp locations using a series ofdifferent paradigms, albeit not consistently (Pang and Fowler,1994). ERP alterations can nevertheless be used as indicators ofphysical, perceptual and semantic changes in the environment thatconstitute physiological indexes of changes in cognition (Kutasand Dale, 1997). The ERP effects of lorazepam have not beencompared to that of other benzodiazepines so it is possible thatthey may help characterize specific changes brought about by thisdrug that might relate to its effects on indirect memory tasks.

Probably the most studied ERP is the P3, or P300, a positivepeak that occurs approximately 300 ms after stimulus onset andreflects cognitive processing (Polich, 1994; Pooviboonsuk et al.,1996). The exact nature of this type of processing, as well as thephysiological processes engendered in the generation of the P3, areunknown (Kügler et al., 1993). It has nevertheless been suggestedthat the P3 reflects updating of working memory, cognitive closure,and transfer of information to consciousness (Kutas and Dale,1997). Earlier exogenous components, N1 and P2, have beenlinked to early feature analysis and attentional gating, while N2 hasbeen associated to stimulus discrimination (Iragui et al., 1993).

The aim of the present study was to compare mnemonic,psychomotor and subjective effects of two benzodiazepinecompounds with the same affinity to the benzodiazepine bindingsite: lorazepam, the only benzodiazepine consistently shown toimpair indirect measures of memory, and flunitrazepam, a drugwith classical benzodiazepine effects (Ingum et al., 1993a,b;Pompéia et al., 1996a,b). If lorazepam’s unique impairment ofimplicit memory is attributable to its high potency, it is to beexpected that an equipotent drug such as flunitrazepam should leadto similar effects. In addition to behavioural parameters, ERPalterations of these benzodiazepines were used as physiologicalindexes of changes in cognition.

Methods

SubjectsSixty physically healthy, native Portuguese speaking volunteers (30men, 30 women), aged 18–36 years (mean ± SD: 24.8 ± 5.0 years),with average body mass index (weight/height2: 22.0 ± 2.1 kg/m2),more than 12 years of schooling, and normal trait anxiety (STAI,37.4 ± 7.9). STAI cut-off scores were those higher thanmean + 1 SD of norms for Brazilian University students(Gorenstein et al., 1995). Participants met the usual exclusioncriteria for clinical trials (e.g. pregnancy, allergy, chronic clinical orpsychiatric disorders), had no history of drug abuse or heavy

S. POMPÉIA et al.: DOUBLE-DISSOCIATION BEHAVIOURAL AND EVENT-RELATED POTENTIAL EFFECTS 289

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alcohol drinking, consumed less than five units of alcohol perweek, did not smoke or regularly use drugs of abuse such ascannabis and cocaine and were on no medication at the time of thestudy.

ProcedureThis was an independent group-design study of single oral doses ofbenzodiazepines. Subjects were randomly allocated, apart frombalancing by sex, to one of five treatments: placebo (P), 2.0 mg oflorazepam (L) and 1.0 mg, 0.8 mg and 0.6 mg of flunitrazepam (F).Potency of both benzodiazepine are equivalent [Ki: Möhler andOkada (1978); Arendt et al. (1987); IC50: Möhler and Okada(1977); Müller (1987)]. The Ethics Committee of the institution atwhich the experiment was conducted (UNIFESP) approved theprotocol and all subjects signed informed consent forms. Subjectswere instructed to abstain from alcohol or other drugs for 24 hbefore and after the experiment. In the morning of the testing day,subjects ingested a light breakfast (with their usual intake ofcaffeine) provided at the laboratory. Participants were tested beforetreatment and again approximately at theoretical peak plasmaconcentration of drugs, 90 min for F (Mattila and Larni, 1980) and120 min for L (Ameer and Greenblatt, 1981). Subjects werefamiliarized with all pre-treatment tests before performance wasassessed. Different versions of all tests were counterbalancedacross subjects, treatments and experimental sessions (pre- andpost-treatment). All tests were carried out in the same order (Table1). The stem-completion tasks were assessed only in the post-treatment session so as to diminish the possibility that explicitstrategies be employed in the indirect task (Buffett-Jerrott et al.,1998). Counterbalancing for the latter measures occurred onlywithin subjects and treatments.

Scores on delayed recall of prose (see below) were used todetermine which of the three doses of F was equipotent to 2.0 mgof L in terms of episodic memory impairment because this was thekey construct of interest in the present study and becausepsychomotor and subjective changes have been reported to reachceiling effects at relatively low doses (Rall, 1990; Pompéia et al.,1996a). Stem-completion performance was not used to equatedoses because impairment in this task could be unique to L.

TreatmentFormulated in identical capsules and administered orally in thefollowing double-blind method: subjects in the F treatmentsreceived a placebo capsule at 09.30 h and another containing thedrug at 10.00 h; those in the L treatment received the activesubstance at 09.30 h, followed by placebo 30 min later; the Ptreatment ingested two placebo capsules at the same times. Post-treatment measures began with ERPs at 11.00 h (30 min beforetheoretical peak), electrodes were removed and the behaviouralmeasures were conducted at 11.30 h (at peak plasma concentrationof L and F). ERPs were tested just before peak because the majoreffects under investigation in the present study werebenzodiazepine-induced memory changes, which where thereforeconducted at peak.

Test batteryPsychomotor/attention and perceptual tasks(i) Cancellation Test (CT; Bond and Lader, 1972): a paper andpencil measure of focused attention at speed, scored for the timetaken to cross out the number 4 which appeared at a frequency of

40 in 400 random digits. One second was added to the score foreach error of omission (no errors of commission were observed).

(ii) Digit-Symbol Substitution Test (DSST; Wechsler, 1955): apaper and pencil test involving coding skills and psychomotorability. Subjects were required to substitute digits for symbols in90 s. Scores were the total number of correct substitutions.

(ii) Visual Perception (PSSCogRehab. v.95, PsychologicalSoftware Services, Inc., Indianapolis, IN, USA): a perceptually andattentionally demanding task. Subjects watched the computerscreen as random dots appeared sequentially to form one of 16geometrical shapes displayed on a grid on the monitor. Subjectswere required to select, using the mouse, the shape that was beingformed as quickly as possible. Scores were mean time taken tochoose each of 15 shapes, and the number of mistakes.

Subjective ratings(i) Visual-Analogue Scales (VAS): rating was performed bymarking a point in a line of 100 mm which represented the fullrange of a particular dimension. Subjects (SUBJ) and theexperimenter (EXP) rated subjects’ arousal (from ‘very alert’ to‘very sleepy’), competence (competent/incompetent) and attention(attentive/distracted), as well as the nature of treatment (placebo/drug). A VAS scale, through which subjects indicated familiaritywith the computer (‘could not be higher’ to ‘could not be lower’),was also included pre-treatment. Scales were analysed individually.

(ii) The State-Trait Anxiety Inventory (STAI; Spielberger et al.,1970; Biaggio and Natalício, 1979): this consists of two scaleslisting 20 statements related to symptoms or feelings of anxiety.Each statement is rated by subjects from 1–4 according to theintensity or frequency at which the feelings are experienced.Scores are the addition of points of each scale. Adjustments aremade for the statements that are reverse coded. The Trait-anxietyscale, measured only pre-treatment, evaluates individual pronenessto anxiety as a personal trait and was used to exclude volunteerswith high trait anxiety. The State scale assesses momentaryfeelings of anxiety and was evaluated pre- and post-treatment.

Memory tasks(i) Prose Recall (Correa and Gorenstein, 1988a,b): volunteers wereasked for immediate and delayed (approximately 20 min) spokenrecall of a story of 14 ‘idea items’ presented orally. Scores werebased on the number and precision of items recalled (1 = perfectrecall; 0.5 = partial recall or synonym). Scores on delayed recall of

290 JOURNAL OF PSYCHOPHARMACOLOGY 14(3)

Table 1 Order of testing

Pre-treatment Post-treatment

Immediate recall of prose ERPsVisual perception Perceptual/semantic word

encodingCT CTDSST DSSTDelayed recall of prose Indirect stem-completion Subjective ratings Direct stem-completionERPs Immediate recall of prose

Visual perceptionDelayed recall of prose Subjective ratings

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prose were used to determine which of the doses of F wasequipotent to 2 mg of L.

(ii) Stem-Completion: (1) a pool of 150 neutral, Portuguese words(nouns, adjectives, verbs) with unique three-letter stems were used:(a) 108 five-letter words were organized into nine sets of 12 wordsbalanced according to rank among other completions for eachstem, number of completions per stem (3–7), and chancecompletion (all determined in a pilot study with 70 universitystudents); (b) 36 buffer words belonging to six semantic categoriesgrouped in three 12-word sets (not scored); (c) three other bufferwords to control for primacy and three for recency effects (notscored). Three presentation lists were constructed by combiningthree sets of five-letter-words and one set of categorized words(ordered randomly in each list) plus primacy and recency items.Three recall-lists were compiled to be used in the stem-completiontask. They contained 24 stems that could be completed with wordsseen before (primed words: 12 encoded semantically and 12perceptually; see below), and 12 stems corresponding to wordsseen by other subjects (unprimed words used to determine base-rate completion), all randomly positioned in the lists. (2)Study/Encoding Tasks: words of two presentation lists were shownon a computer screen at the rate of one word every 5 s (font Arialno. 36, uppercase, bold). Subjects were instructed to say each wordaloud and carry out a different task for each list: (a) rate how mucheach word was liked on a 5-point scale (semantic encoding), (b)count the total number of enclosed spaces in the letters of eachwords (e.g. ‘B’ has two enclosed spaces, ‘E’ has none) (perceptualencoding). At study and test subjects responded aloud and theexperimenter recorded their responses. (3) Tests: subjects wereshown stems from two of the three recall lists. Stems werepresented on the computer screen for 15 s each (in the samelettering as at study). For the first list, subjects were instructed tocomplete stems with the first word that came to mind (indirectstem-completion). For the second list, volunteers were asked tocomplete stems with words seen at study (direct stem-completionor cued recall). The dependent variables scored were percentage ofstems completed with target words (those that appeared in theword-lists used) and percentage of stems left blank.

Event Related PotentialsThe equipment used was a Nihon Kolden, model Sigma (MEB5508). The auditory oddball paradigm was employed, thecommonest ERP measure studied to date. Subjects were presentedbinaurally with a series of 800 Hz and 1500 Hz tones at 70 dBrandomly presented at the rate of one tone every 2 s. The hightarget tones were presented at a frequency of 20% and the lowfrequent tones at a rate of 80%. Subjects were required to keeptheir eyes open and press a button with their dominant handwhenever they heard the target stimuli. ERPs were recorded usingelectrodes at midfrontal (FZ), midcentral (CZ) and midparietal(PZ) scalp locations (10–20 international system), and werereferenced to linked earlobes. The electro-oculogram (EOG) wasrecorded from electrodes above the right eyebrow and just lateralto the outer canthus of the left eye. A total of 15 target tones free ofEOG and movement artefacts were averaged and replicated.Signals were amplified, bandpass filtered between 0.1–50 Hz, andrecorded with a bin width of 1 ms. Latency of N1, P2, N2 and P3for rare stimuli and N1 and P2 for frequent stimuli (N2 and P3

seldom appear in response to frequent stimuli) and peak to peakamplitude of N1–P2, P2–N2, N2–P3 for rare stimuli and N1–P2for frequent stimuli were measured at FZ, CZ and PZ individuallyand averaged across the three electrode locations. N1 and N2 wereconsidered the most negative peaks registered after stimulus onsetbetween 50 and 150 ms, and 175–275 ms, respectively; P2 and P3(P3max) were the most positive peaks present between 125 and230 ms, and 250–500 ms (Iragui et al., 1993).

Results

For variables assessed pre- and post-treatment, change scores (post-minus pre-treatment) were used in the analysis when no groupdifferences were found pre-treatment. Non-parametric Kruskall–Wallis tests followed by Mann–Whitney tests were employed whenvariables did not display equality of variances (Bonferroni correc-tions were employed). When equality of variances were found,analysis were conducted using analysis of variance (ANOVAs)followed by Tukey t-tests. The significance level of 5% wasadopted for all statistical comparisons. Variables and comparisonsthat are not cited below did not show significant effects.

No differences among treatments were observed in age, bodymass index, trait anxiety, or familiarity with the computer. Stateanxiety change scores also did not vary between groups. Nodifferences pre-treatment were found in the behavioural (non-ERP)tasks. Two subjects were replaced: one from the 1.0 mgflunitrazepam treatment who could not be kept awake during thepost-treatment session, and one participant who took L anddisplayed intolerance to the drug.

Determining the dose of flunitrazepam that wasequipotent to 2.0 mg of lorazepam in terms of episodicmemoryThe one-way ANOVA on change-scores of delayed recall of prose(Fig. 1) revealed a significant main effect of treatment[F(4,54) = 6.28, p < 0.0004]. Placebo scores were higher thanthose of L (p < 0.004) and 1.0 mg of F (p < 0.0002). The otherdoses of F were not differentiated from P (ps > 0.62). The analysisdescribed below for scores on the remaining behavioural tasks(Table 2) and ERPs therefore involved only P, L and 1.0 mg offlunitrazepam (F1.0 mg).

Psychomotor/attention and perceptual tasksCancellation time [F(2,30) = 7.89, p < 0.002] was faster for P thanL (p < 0.004) and F1.0 mg (p < 0.02). In DSST (H = 19.80,p < 0.0001, d.f. = 2), both active treatments were also impaired inrelation to P (p < 0.0003). By contrast, reaction time of the visualperception task [F(2,33) = 16.83, p < 0.0001] was slower for Lthan F1.0 mg and P (ps < 0.0005).

Subjective ratingsArousal as measured by subjects [F(2,32) = 8.66, p < 0.001] andthe experimenter (H = 14.42, p < 0.00003, d.f. = 2) differentiatedtreatments. Subjects in the P treatment rated less sedation thanthose in the L (p < 0.02) and F1.0 mg (p < 0.002) groups, the sameoccurring for the EXP scale (p < 0.0004). In terms of attention,subjects (H = 7.51, p < 0.02, d.f. = 2) in the F1.0 mg treatment ratedhigher distraction than those in the P group (p < 0.007), while the

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experimenter (H = 16.00, p < 0.0003, d.f. = 2) consideredvolunteers treated with L and F1.0 mg as more distracted than thosewith P (p < 0.002). Only subjects who received treatment F1.0 mgrated worsening of competence (H = 9.84, p < 0.007, d.f. = 2) incomparison to P (p < 0.001). The subjects [F(2,33) = 16.29,p < 0.0001] and the experimenter [F(2,32) = 939.23, p < 0.00001]correctly rated L and F1.0 mg as having drug effects and P as theinactive treatment (ps < 0.0003).

Memory tasksDelayed recall of prose [F(2,32) = 9.90, p < 0.0005] was better forP than both L and F1.0 mg (p < 0.003), while L and F1.0 mg showedthe same effects (p = 0.99) since this task was used earlier todetermine equivalence of the active treatments in terms of episodic

memory effects. No differences between treatments were found onimmediate prose-recall (p > 0.32).

Indirect stem-completionA two-way ANOVA with treatment (L × F1.0 mg × P) and encoding(primed perceptual × primed semantic × unprimed) as factors wasused to analyse the percentage of stems completed with targetwords. Treatment [F(2,33) = 5.56, p < 0.009] and encoding effects[F(2,66) = 34.81, p < 0.000001] were found, but there was nointeraction [F(4,66) = 0.93, p > 0.45]. Subjects in the L group usedless target words than those treated with P (p < 0.007), while thosewho took F1.0 mg were no different from P nor L-treated volunteers(ps > 0.19). Memory was observed in all treatments becauseoverall stems of unprimed words (base-rates) were completed lessoften with target items than stems completed with primed wordsencoded perceptually and semantically (ps < 0.0002). Nodifference between perceptual and semantic encoding were found(p > 0.64).

An additional set of analysis was conducted to furtherinvestigate priming effects. Initially, a one-way ANOVA withtreatment as factor was carried out on base-rate completion. Nodifference was found between groups [F(2,33) = 1.36, p > 0.27] soa two-way ANOVA with encoding as factor (per-ceptual × semantic) was carried out on primed scores only. Thisanalysis revealed a significant main effect of groups[F(2,33) = 4.78, p < 0.02], L again showing impairment in relationto P (p < 0.02), while none of the other contrasts approachedsignificance (ps > 0.23). No effect of encoding or interaction wereobserved (ps > 0.28). A one-way ANOVA with treatment as factor

292 JOURNAL OF PSYCHOPHARMACOLOGY 14(3)

Table 2 Pre- and post-treatment scores (mean ± SD) per treatment [2.0 mg of lorazepam (L), 1.0 mg of flunitrazepam (F), and placebo (P)]on behavioural tasks.

L F P

Variable Pre Post Pre Post Pre Post

Psychomotor/perception/attentionCT (s)a 72 ± 16 89 ± 23 80 ± 19 94 ± 26 69 ± 14 68 ± 18DSST (no.)a 65 ± 8 57 ± 22 60 ± 6 52 ± 9 67 ± 11 70 ± 11Visual perception (s)a 8 ± 2 10 ± 2 7 ± 1 7 ± 2 7 ± 1 7 ± 1

VAS (mm)b

EXP (alert × drowsy)a 26 ± 29 74 ± 25 28 ± 23 70 ± 25 28 ± 22 21 ± 14SUBJ (alert × drowsy)a 34 ± 19 64 ± 19 27 ± 19 66 ± 24 27 ± 15 30 ± 21EXP (attentive × distracted)a 17 ± 19 56 ± 40 24 ± 26 34 ± 31 14 ± 13 13 ± 13SUBJ (attentive × distracted)a 35 ± 14 54 ± 24 23 ± 16 56 ± 25 24 ± 21 28 ± 25EXP (incomp. × competent) 90 ± 7 79 ± 25 88 ± 6 84 ± 13 86 ± 6 89 ± 5SUBJ (incomp. v competent)a 74 ± 21 58 ± 23 80 ± 14 54 ± 29 82 ± 12 83 ± 14EXPc (placebo × drug)a 98 ± 7 97 ± 8 0 ± 1SUBJc (placebo × drug)a 84 ± 15 76 ± 28 30 ± 29

MemoryImmediate recall of prose (no.) 8.9 ± 1.8 6.9 ± 2.4 8.6 ± 2.1 6.7 ± 1.6 9.1 ± 2.3 8.6 ± 1.5Delayed recall of prose (no.)* 7.6 ± 1.6 2.1 ± 2.6 7.5 ± 1.7 1.9 ± 2.7 8.2 ± 2.3 7.3 ± 1.6Stem-completionc

Indirect (% perceptual) 48 ± 15 56 ± 18 61 ± 11Indirect (% semantic) 51 ± 12 54 ± 15 68 ± 11Indirect (% base-rates) 29 ± 16 37 ± 13 37 ± 15Indirect (% total left blank) 42 ± 25 22 ± 16 27 ± 20Direct (% perceptual) 43 ± 15 53 ± 12 58 ± 17Direct (% semantic) 54 ± 15 63 ± 16 76 ± 17Direct (% base-rates) 35 ± 14 38 ± 11 27 ± 20Direct (% total left blank) 54 ± 41 19 ± 22 22 ± 19

aVariables that showed significant treatment effects.bVAS (visual-analogue scales): smaller scores relate to first symptom (e.g. alert), higher scores to lastsymptom (e.g. drowsy). cVariables measured only post-treatment; see text for statistical effects of stem-completion tasks.

Figure 1 Change scores (post- minus pre-treatment; mean ± SE) pertreatment [lorazepam (L), flunitrazepam (F), and placebo (P)] ofnumber of items correctly recalled on delayed recall of prose.*p < 0.004.

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was also conducted on the total percentage of stems left blank.Groups tended to differ [F(2,33) = 3.05, p = 0.06], L-treatedsubjects having completed less stems than F (p = 0.06). Becausethe smaller number of completions in the L group could beenhancing a drug effect in this task, an ANCOVA was carried outusing percentage of scores left blank as a covariate in the analysisfor primed scores. The pattern of effects was unchanged.

Direct stem-completionThe effects observed for this task were essentially the same as forthe indirect task except that semantic encoding also led to morecompletions with primed words than perceptual learning: a two-way ANOVA with treatment (L × F1.0 mg × P) and encoding(primed perceptual × primed semantic × unprimed) as factors wasused to analyse the percentage of stems completed with targetwords. Treatment [F(2,33) = 6.06, p < 0.006] and encoding effects[F(2,66) = 37.81, p < 0.000001] were found, but there was nointeraction [F(4,66) = 1.14, p < 0.34]. L used less target words thanP (p < 0.004), while F1.0 mg was no different from P nor L(ps > 0.21). Memory was observed in all treatments because,overall, stems of unprimed words were completed less often withtarget items than with those encoded perceptually and semantically(ps < 0.0003). Also, semantic encoding led to more completionswith target words than perceptual learning (p < 0.003). A one-wayANOVA with treatment as factor was also conducted on the totalpercentage of stems left blank. Groups differed [F(2,33) = 5.56,p = 0.009], L-treated subjects having completed less stems than Fand P (p < 0.03), so an ANCOVA was carried out using percentageof scores left blank as a covariate. The pattern of effects was againunchanged. The same additional set of analysis used for indirectstem-completion was conducted to further investigate memoryeffects. No difference in base-rate completion was found betweengroups [F(2,33) = 1.03, p = 0.37]. The two-way ANOVA withtreatment and encoding (perceptual × semantic) as factorsconducted on primed scores revealed a significant main effect oftreatment [F(2,33) = 7.11, p < 0.03], encoding [F(1,33) = 17.33,p < 0.0003], and no interaction (p > 0.56). L again impairedcompletions in relation to P (p < 0.002) while there were nodifferences for other contrasts (F = L, p = 0.14; F = P, p = 0.18).Semantic encoding led to more completions than perceptuallearning (p < 0.0004). Covarying stems left blank did not changeresults.

Direct versus indirect stem-completionThe three-way ANOVA with treatment (L × F1.0 mg × P), encoding(primed perceptual × primed semantic × unprimed) and test(indirect × direct) as factors revealed a main effect of treatment[F(2,33) = 11.69, p < 0.0002], encoding [F(2,66) = 60.92,p < 0.00001] and interaction between test and encoding[F(2,66) = 3.64, p = 0.03]. L impaired performance in relation to Fand P (p < 0.05) and subjects in the F1.0 mg group tended to beimpaired in relation to those who took P (p = 0.06). In terms of theinteraction, no difference between tests was found for indirect anddirect base-rates (p = 0.56) and these were smaller than primedperceptual and semantic scores in both tasks (ps < 0.0007). Also,semantic encoding in the direct task led to more primedcompletions than perceptual learning in both the indirect (p = 0.03)and the direct tasks (p < 0.0005).

ANCOVAs were used to covary measures which could indicatesedation (DSST, CT and subject-rated subjective sedation) from

performance on delayed recall of prose and from the initialseparate direct and indirect stem-completion analysis. Results wereunchanged. In order to investigate whether L-induced mnemoniceffects were related to visual perception, reaction time on thevisual perception task was also used as a covariate from scores inmemory tests that showed treatment effects. For both stem-completion tasks this led to the disappearance of treatment effects(indirect: [F(2,32) = 1.48, p > 0.24]; direct: [F(2,33) = 1.62,p > 0.21]). Encoding effects were unchanged and no interactionbetween encoding and treatments was observed. By contrast,results on delayed recall of prose were unaltered.

ERPs (Table 3)The only measures that showed differences pre-treatment were N1(FZ) for rare stimuli and P2 (PZ) for frequent stimuli, so pre-treatment scores were covaried from change scores in thesevariables. In general, N1 and P3 latencies referent to rare stimulishowed treatment effects, F1.0 mg displaying higher effects thanboth P and L in various cases (Table 4), while no differences wereobserved for frequent stimuli: N1 (FZ) [F(2,32) = 6.61, p < 0.004]and P3 (CZ) (H = 18.89, p < 0.0002, d.f. = 2) latencies were shorterfor P than both L and F1.0 mg (ps < 0.006), and for L than F1.0 mg(ps < 0.04). N1 (CZ) (F2,33 = 6.59; p < 0.004) and N2 (PZ)(H = 11.73, p < 0.003, d.f. = 2) latencies were higher for F1.0 mgthan L (ps < 0.04) and P (ps < 0.003). P3 (FZ) (H = 16.52,p < 0.0004, d.f. = 2) and P3 (PZ) latencies (H = 16.00, p < 0.00004,d.f. = 2) were shorter for P than L and F1.0 mg (ps < 0.006). N2 (FZ)(H = 7.79, p < 0.02, d.f. = 2) and N1 (PZ) [F(2,33) = 5.59,p < 0.008] latencies were only higher for F1.0 mg than P (p < 0.009).N2 (CZ) (H = 6.47, p < 0.04, d.f. = 2) did not differentiatetreatments in the post-hoc analysis. In terms of averaged scoresamong locations, N1 [F(2,33) = 9.17, p < 0.0007] and P3[H = 18.08, p < 0.0002, d.f. = 2] latencies were prolonged forF1.0 mg in relation to P (ps < 0.0007) and L (ps < 0.03); N2 latency(H = 7.47, p < 0.03, d.f. = 2) was also longer for F1.0 mg than P(p < 0.02). In terms of amplitude, treatment effects occurred forP2–N2 at CZ [F(2,33) = 4.43, p < 0.02], L and F1.0 mg beingsmaller than P (p < 0.04), and for N1–P2 at PZ [F(2,33) = 3.54,p < 0.05], for which L was higher than F1.0 mg (p < 0.05). Averagedresults showed treatment effects only for N2–P3 [F(2,33) = 3.54,p < 0.05], L having shown lower amplitude than P (p < 0.02).Grand averages were obtained in order to illustrate waves (Fig. 2)and were not used for analysis.

DiscussionTable 4 summarizes the main findings of the present study andshows that 2.0 mg of L and 1.0 mg of F led to equivalent effects ontasks that tap a wide spectrum of cognitive functions. This tablealso shows a double-dissociation between benzodiazepines: visualperception was more impaired by L while ERP latency changeswere more pronounced for F1.0 mg. The similarity of effects on amyriad of tasks and this double-dissociation lend support to theassertion that these doses are equivalent in terms of cognitiveimpairment (see Duka et al., 1996). In other words, if only one ofthe doses had led to higher impairment in one or more tasks, itwould follow that these tasks were more sensitive to differencesbetween Bzs doses and that the dose of the other medication had tobe increased so that similar effects were established. However,because the doses of L and F showed higher effects in two distinctperformance measures, test sensitivity cannot explain the

S. POMPÉIA et al.: DOUBLE-DISSOCIATION BEHAVIOURAL AND EVENT-RELATED POTENTIAL EFFECTS 293

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difference in effects, suggesting that these tasks reflect specificcentral effects of L and F and that no better match of doses can beachieved.

Corroborating findings in the literature on benzodiazepineseffects, L and F1.0 mg similarly impaired psychomotor performance

(Curran, 1991), and did not alter short-term memory as measuredby immediate recall of prose (Pompéia et al., 1996a). Bothbenzodiazepines also produced clear and equivalent impairment ofauditory delayed recall of prose, corroborating the anterogradeepisodic memory effect of benzodiazepines (Curran et al., 1987;

294 JOURNAL OF PSYCHOPHARMACOLOGY 14(3)

Table 3 Pre- and post-treatment scores (mean ± SD) per treatment [2.0 mg of lorazepam (L), 1.0 mg of flunitrazepam (F), and placebo (P)]on Event-Related Potential (ERP) latencies according to scalp locations and type of stimuli (rare or frequent-freq.) (*latencies withsignificant treatment effects).

L F PLocation(stimuli) Potential Pre Post Pre Post Pre Post

FZ (rare) N1* 95 ± 10 97 ± 8 95 ± 14 112 ± 21 111 ± 17 101 ± 13P2 167 ± 14 172 ± 21 174 ± 16 184 ± 22 178 ± 11 180 ± 20N2* 215 ± 18 236 ± 42 218 ± 18 268 ± 55 221 ± 20 227 ± 26P3* 320 ± 19 337 ± 29 317 ± 25 398 ± 56 327 ± 18 327 ± 19

FZ (freq.) N1 106 ± 19 97 ± 13 104 ± 19 105 ± 19 108 ± 16 108 ± 17P2 190 ± 28 188 ± 30 179 ± 18 176 ± 12 176 ± 18 182 ± 20

CZ (rare) N1* 98 ± 13 95 ± 9 98 ± 16 108 ± 21 105 ± 15 96 ± 11P2 168 ± 12 168 ± 21 167 ± 15 186 ± 22 168 ± 12 173 ± 22N2 210 ± 15 218 ± 18 206 ± 14 262 ± 61 210 ± 13 218 ± 23P3* 318 ± 21 336 ± 28 315 ± 26 398 ± 55 327 ± 17 327 ± 20

CZ (freq.) N1 107 ± 18 100 ± 15 100 ± 14 100 ± 20 105 ± 16 106 ± 14P2 194 ± 23 185 ± 26 178 ± 18 171 ± 11 174 ± 12 182 ± 20

PZ (rare) N1* 98 ± 11 95 ± 9 98 ± 17 108 ± 25 104 ± 15 95 ± 10P2 168 ± 16 173 ± 23 170 ± 14 192 ± 24 168 ± 13 172 ± 23N2* 206 ± 14 228 ± 37 205 ± 16 260 ± 51 214 ± 19 216 ± 21P3* 317 ± 22 336 ± 29 301 ± 45 397 ± 54 328 ± 18 328 ± 22

PZ (freq.) N1 105 ± 18 98 ± 11 94 ± 15 93 ± 15 99 ± 12 105 ± 18P2 195 ± 19 190 ± 26 174 ± 15 173 ± 13 174 ± 14 183 ± 22

Average N1* 97 ± 11 96 ± 8 97 ± 16 110 ± 22 107 ± 15 97 ± 10(rare) P2 167 ± 13 171 ± 21 170 ± 14 187 ± 21 171 ± 11 175 ± 22

N2* 210 ± 15 227 ± 31 210 ± 13 263 ± 54 215 ± 16 220 ± 22P3* 318 ± 20 336 ± 28 311 ± 27 368 ± 55 327 ± 18 327 ± 20

Average N1 106 ± 18 98 ± 11 99 ± 13 99 ± 16 104 ± 14 106 ± 15(freq.) P2 193 ± 22 188 ± 26 177 ± 17 173 ± 10 175 ± 14 182 ± 20

Figure 2 Grand average Event Related Potentials (ERPs) pre-treatment (thin lines) and post-treatment (thick lines) at electrode locations FZ, CZ, PZ andelectro-oculogram (EOG).

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Curran and Gorenstein, 1993; Pompéia et al., 1996b; Fluck et al.,1998). In addition, a broadly similar pattern of effects emerged onsubjective measures of sedation and in judging whether treatmentsconsisted of placebo or drug. However, groups differed in terms ofsubjective measures of attention and feelings of competence. L-treated subjects were unable to judge their own impairment,showing the disruption of metacognitive functions that has beendescribed for triazolam (see Weingartner et al., 1992).

In the direct and indirect stem-completion tasks, all treatmentsshowed memory effects by completing more stems with primedwords than by chance. However, in contrast to the similarity ofeffects of L and F1.0 mg aforementioned, only L impaired indirectstem-completion when compared to P, suggesting the reduction ofpriming that has been repeatedly reported for L (Curran andGorenstein, 1993; Legrand et al., 1995; Stewart et al., 1996;Buffett-Jerrott et al., 1998). Considering the results of applicationof the ‘Retrieval Intentionality Criterion’ (Schacter et al., 1989),contamination by episodic memory cannot explain the effect on theindirect task because semantic encoding, known to enhanceepisodic memory and leave implicit memory unchanged, did notimprove performance on this task for any of the treatments, while itdid so on the direct version of this task for all groups. Similar resultswere reported by Bishop and Curran (1995) in a study using L.

Comparisons of the different effects of both benzodiazepines onthe two direct memory tasks used (delayed recall of prose anddirect stem-completion) can only be made if differences betweenthem are considered. One of these differences relates to taskdemands. Delayed recall of prose is a free recall task that imposesmore demands at retrieval because no cues are presented, such as

stems which can facilitate recall of previously learned information.As benzodiazepine impairment seems to be smaller under lowertask demands (see Pompéia et al., 1996b), a more impairing doseof a benzodiazepines should have led to larger effects in this test.The similarity of effects on delayed recall of prose for both drugssuggests that 1.0 mg of flunitrazepam is in fact comparable to2.0 mg of lorazepam, supporting data on psychomotor andsubjective alterations of these doses. Because potency of both Fand L are equivalent, the high potency of L does not explain itsalleged implicit memory effect evaluated through indirect stem-completion, nor its impairment of the direct version of this task.

Another important aspect that distinguishes delayed recall ofprose from stem-completion is that the former is an auditory taskwhile the latter involves visual processing. We suggest that thischaracteristic of the stem-completion task may be one of thedeterminants of the L-induced atypical memory effects for threereasons: only L impaired memory as measured by both the directand indirect versions of this task; the only other behaviouralvariable that was more affected by L was the reaction time inidentifying shapes; L induced smaller latency auditory ERPchanges than F1.0 mg as discussed below. When time to identifyshapes was covaried from direct and indirect stem-completion, Leffects were abolished, suggesting that these tasks involve similarcognitive processes, possibly changes in visual perception. It isunlikely that the motor component of the visual perception taskwas responsible for this effect because covarying thesedative/attentional effects of the benzodiazepines, as measured bythe CT, the DSST, and the subjects’ subjective measure of sedationdid not alter the pattern of effects on the stem-completion tasks.

S. POMPÉIA et al.: DOUBLE-DISSOCIATION BEHAVIOURAL AND EVENT-RELATED POTENTIAL EFFECTS 295

Table 4 Comparison of the significant effects of 2.0 mg of lorazepam (L), 1.0 mg of flunitrazepam (F), and placebo (P) on variables thatshowed differences among treatments (*p < 0.05; **p < 0.01; ***p < 0.001.

Type of task Variable (L&F) ≠ P L ≠ F L or F ≠ P

Psychomotor CT (s) (L&F) > P*DSST (no.) (L&F) < P***Visual perception (s) L > F*** L > P***

Memory Delayed recall of prose (no.) (L&F) < P**Indirect stem-compl. (primed%) L < P**Direct stem-compl. (primed%) L < P**

VASa EXP (alert × drowsy) (L&F) > P***SUBJ (alert × drowsy) (L&F) > P*EXP (attentive × distracted) (L&F) > P**SUBJ (attentive × distracted) F > P**SUBJ (competent × incompetent) F > P***EXP (placebo × drug) (L&F) > P***SUBJ (placebo × drug) (L&F) > P***

ERP latency N1 at FZ (ms) (L&F) > P*** F > L*(rare stimuli) N2 at FZ (ms) F > P**

P3 at FZ (ms) (L&F) > P**N1 at CZ (ms) F > L* F > P**P3 at CZ (ms) (L&F) > P*** F > L**N1 at PZ (ms) F > P**N2 at PZ (ms) F > L** F > P**P3 at PZ (ms) (L&F) > P**N1 (averaged; ms) F > L* F > P***N2 (averaged; ms)P3 (averaged; ms) F > L*** F > P*

ERP amplitude P2-N2 (µV) (L&F) < P* F > P***N1-P2 (µV) F < L*N2-P3 (averaged; µV) L < P*

aVAS (visual-analogue scales): smaller scores relate to first symptom (e.g. alert), higher scores to last symptom (e.g. drowsy)

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Furthermore, the lack of alteration of the long term memory effectsafter covarying these same sedative/attentional measures seems tocorroborate that mnemonic alterations caused by benzodiazepinesare not exclusively secondary to sedation (Curran et al., 1998).

The possibility that L alters visual perception is supported bythe finding that it impairs the identification of pictures on the basisof contour fragments, exhibiting central effects that are notobservable for other benzodiazepines (Wagemans et al., 1998),possibly through reduction of perceptual processes at encodingand/or retrieval (Sellal et al., 1992; Vidailhet et al., 1994;Wagemans et al., 1998). Peripheral changes, such as alterations invisual acuity, do not seem to be involved (Giersch et al., 1996).Reduction in visual perception could increase time to identifyshapes and decreased the percentage of completions with primedwords because performance in stem-completion tasks relies onperceptual similarity between stems and stimuli encoded at study(Blaxton, 1989). In fact, it is not surprising that a similar effect forL was found in the stem-completion tasks and in shapeidentification if one considers that they may rely on the sameperceptual representation system (Tulving and Schacter, 1990).Recent findings suggest that the L-induced impairment of thissystem may not be exclusive to visual processing and/or that L mayimpair access to lexical units (Vidailhet et al., 1999), although thisdoes not explain the small auditory ERP latency changes withrespect to L discussed below. Whatever the mechanisms involvedin the stem-completion impairment by L, they seem to be unique tothis benzodiazepine. Further studies are needed to investigate thenature of the perceptual changes induced by this drug.

It is difficult to establish whether L affects perception at studyand/or at test. Our data suggests that the latter hypothesis becauseof the lack of interaction between treatments and LOP in both stem-completion tasks. This confirms that benzodiazepines, including L,impair performance independently of the level at which words areinitially encoded (Curran et al., 1988; Curran et al., 1993).

The reason why L may have an atypical effect on indirect stem-completion and/or perception has received scant attention. Thepharmacokinetics of this drug cannot be differentiated from thoseof other benzodiazepines (Greenblatt and Shader, 1987), nor doesL have a specific binding profile to distinct benzodiazepinereceptor subtypes (Piercey et al., 1991; Miller et al., 1992).However, L seems to exhibit slower receptor association–dissociation rate constants than other benzodiazepines (Jack et al.,1983; Ellinwood et al., 1985). It is possible that these constants aremore important in determining memory and/or perceptualalterations of benzodiazepines than affinity itself.

In contrast to the slightly more impairing overt behaviouraleffects of L, F1.0 mg prolonged N1 and P3 latencies overall to agreater degree, potentials that are related to attentional andcognitive processing, respectively. Considering that ERP measureswere conducted just before peak-plasma concentration, it could besuggested that the smaller ERP latency changes after L were due toloss of peak effect (Buffett-Jerrott et al., 1998). However, webelieve this is unlikely because comparably small (Samra et al.,1988; Pooviboonsuk et al., 1996; Curran et al., 1998) or no(Nichols and Martin, 1993) L-induced P3 latency changes at peakin normal volunteers have been reported. Also, differences betweendrugs in terms of acute tolerance cannot explain these results as Lseems to lead to practically no development of acute tolerancecompared to other compounds, including alprazolam, which hassimilar receptor affinity to that of L (Ellinwood et al., 1985, 1987).

Unfortunately, amplitude changes did not contribute to thecharacterization of the effects of the benzodiazepines because itcould not be established which of the peaks, in the peak to peakamplitudes measured, were altered, and in which direction(increased or decreased). Peak to baseline measures may constitutea better means of characterizing drug effects.

Differences in ERP profiles for L and F1.0 mg may relate to thenature of the tasks under investigation, whether auditory orvisually based. L may indeed lead to atypical effects primarily invisual tasks as discussed above. Alternatively, these differencesmay relate to the clinical profile of the drug, i.e. whethertranquillizer (lorazepam) or hypnotic (flunitrazepam). The relation-ship between sedation and ERP components is still unclear. Urataet al. (1996) showed P3 latency increases after small doses oftriazolam that did not induce sedation. Pang and Fowler (1994)also concluded that increases in P3 latency after triazolam did notsupport the sedation view of benzodiazepine effects on stageprocessing. Prolongation of N1 and P3 latencies have also beenascribed to sedative effects of intravenous lorazepam, which leadsto much higher sedation than oral doses, but only when theyoccurred for both frequent and rare stimuli (Samra et al., 1988),which was not the case in the present study. Still, if ERP latencychanges in fact reflect differences in profile between hypnotics andtranquillizers, they might prove useful in characterizing the effectsof new benzodiazepines.

In summary, L decreased performance in relation to F1.0 mg andP in time to identify fragmented shapes. L also impairedperformance on direct and indirect stem-completion in comparisonto P, but this effect was abolished when time to identify fragmentedshapes was used as a covariate. By contrast, F1.0 mg prolongedauditory ERP latencies (N1 and P3) when compared to both P andL. Impairment by L seems to increase when visual processing isinvolved. This impairment does not seem to be related to the highaffinity of L to the benzodiazepine receptor. Further research intoatypical effects of L on perception may lead to a more appropriateclinical application of this benzodiazepine and a betterunderstanding of cognitive phenomena.

Acknowledgements

We thank AFIP and FAPESP for financial support, and Roche andWyeth-Whitehall for supplying the benzodiazepines.

Address for correspondence

O. F. A. BuenoDepartamento de PsicobiologiaUNIFESPR. Napoleão de Barros, 925CEP: 04024–002São Paulo, BrazilEmail: ofabueno@psicobio.epm.br

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