Psychopharmacology (2007) 192:425440 DOI 10.

1007/s00213-007-0726-y

ORIGINAL INVESTIGATION

A triazolam/amphetamine doseeffect interaction study: dissociation of effects on memory versus arousal

Miriam Z. Mintzer & Roland R. Griffiths

Received: 5 December 2006 / Accepted: 24 January 2007 / Published online: 7 March 2007 # Springer-Verlag 2007

Abstract Rationale In addition to producing robust memory impairment, benzodiazepines also induce marked sedation. Thus, it is possible that the observed amnestic effects are secondary to more global sedative effects and do not reflect a specific primary benzodiazepine effect on memory mechanisms. Objective The objective was to use the nonspecific stimulant d-amphetamine to dissociate the sedative and memoryimpairing effects of the benzodiazepine triazolam. Materials and methods Single oral doses of placebo, triazolam alone (0.25, 0.50 mg/70 kg), d-amphetamine sulfate alone (20, 30 mg/70 kg), and triazolam (0.25, 0.50 mg/70 kg) and d-amphetamine sulfate (20, 30 mg/ 70 kg) conjointly (at all dose combinations) were administered to 18 healthy adult participants across nine sessions in a double-blind, staggered-dosing, crossover design. In addition to standard data analyses, analyses were also conducted on z-score standardized data, enabling effects to be directly compared across measures. Results Relative to the sedative measures, the memory measures generally exhibited a pattern of less reversal of triazolams effects by d-amphetamine. The memory measures ranged in degree of reversal such that the most
M. Z. Mintzer (*) : R. R. Griffiths Department of Psychiatry and Behavioral Sciences, Behavioral Biology Research Center, Johns Hopkins University School of Medicine, 5510 Nathan Shock Drive, Baltimore, MD 21224, USA e-mail: mmintzer@jhmi.edu R. R. Griffiths Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA

reversal was observed for reaction time on the n-back working memory task, and the least reversal was observed for accuracy on the Sternberg working memory task, with most measures showing an overall pattern of partial reversal. Conclusions Benzodiazepines have specific effects on memory that are not merely a by-product of the drugs sedative effects, and the degree to which sedative effects contribute to the amnestic effects varies as a function of the particular memory process being assessed. Keywords Triazolam . Amphetamine . Memory . Arousal . Sedative It is well documented that benzodiazepines (e.g., diazepam, Valium; lorazepam, Ativan; and triazolam, Halcion) induce temporary amnesia when administered acutely to healthy volunteers (for reviews, see Curran 1991, 2000; Ghoneim 2004a,b). Like neuropsychological studies of brain-damaged patients, which have played a critical role in advancing the understanding of normal and abnormal memory mechanisms, the investigation of benzodiazepineinduced amnesia can also be a powerful tool for elucidating memory mechanisms. However, in addition to amnesia, benzodiazepines also induce marked sedation as reflected in changes in observer and subjective ratings of arousal and impaired psychomotor performance (for reviews, see Hollister et al. 1993; Woods et al. 1992). Thus, in order for benzodiazepines to be used to advance theoretical understanding of memory mechanisms, it is important to be able to separate the amnestic effects from the sedative effects. A variety of strategies can be adopted to differentiate the amnestic effects of benzodiazepines (or other sedative drugs) from their sedative effects (cf. Curran 1991, 2000;

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Ghoneim 2004b). First, the effects can be dissociated statistically by covarying measures of sedation from measures of memory; results suggest that drug effects on memory typically are somewhat reduced but remain significant once effects on sedation are partialled out (cf. Curran 1991). Second, it can be demonstrated that different drugs produce different patterns of effects on sedation vs memory. For example, it has been shown that at doses of lorazepam and the neuroleptic chlorpromazine (Green et al. 1996) or the antihistamine diphenhydramine (Curran et al. 1998) that produced similar effects on measures of sedation, only lorazepam affected measures of memory. Likewise, using relative potency analyses, Kirk et al. (1990) demonstrated that triazolam showed relatively greater potency than pentobarbital on measures of memory than on measures of sedation. Using pharmacokineticpharmacodynamic (PKPD) modeling techniques, Veselis et al. (1997) showed that sedative drugs (midazolam [a benzodiazepine], propofol, thiopental, and fentanyl) varied in their relative effects on sedation vs memory. Third, it can be demonstrated that tolerance over the course of repeated benzodiazepine administration develops differentially to effects on sedation vs memory. The general conclusion from these studies is that tolerance develops to the sedative effects before the amnestic effects, and that complete tolerance may not develop to the amnestic effects even after years of chronic use (Curran et al. 1994; Ghoneim et al. 1981; Tata et al. 1994). Fourth, it can be demonstrated that the doseresponse and/or timecourse functions of the effects on sedation vs memory have different characteristics (e.g., Blin et al. 2001; Smirne et al. 1989; Weingartner et al. 1995). Using PKPD modeling, Blin et al. (2001) dissociated the sedative and amnestic effects of lorazepam based on the median effective concentration (EC50) needed for each effect. Fifth, sedative and amnestic effects can be dissociated through electrophysiological or neuroimaging techniques. Curran et al. (1998) and Veselis et al. (2001) both provided evidence that the sedative and amnestic effects of benzodiazepines are associated with different event-related potential components. To our knowledge, no studies have attempted to differentiate the sedative and amnestic effects of a benzodiazepine using neuroimaging. Finally, it can be demonstrated that another compound selectively reverses benzodiazepines effects on one function but not the other. Several studies have attempted to differentiate effects of benzodiazepines using the benzodiazepine-receptor specific antagonist flumazenil (Curran and Birch 1991; Hommer et al. 1993; Preston et al. 1989). Results of these studies are mixed, and interpretation is complicated by the fact that flumazenil acts at benzodiazepine receptors, which are presumed to at least partially mediate both the sedative and amnestic effects of benzodiazepines, and by evidence that flumazenil produces its own

weak agonist effects including significant memory impairment (e.g., Bishop and Curran 1995; Neave et al. 2000). In the present study, we used this final strategy to dissociate the sedative and amnestic effects of the benzodiazepine hypnotic triazolam by administering the nonspecific stimulant dextroamphetamine (d-amphetamine). d-Amphetamine acts as an indirect catecholaminergic agonist by facilitating the action of dopamine and norepinephrine (for a review, see Solanto 1998). As reviewed above, the results of previous studies provide evidence that the amnestic and sedative effects of benzodiazepines can be dissociated. However, they do not allow conclusions to be drawn about possible differences among memory processes in the relative contribution of sedative effects to benzodiazepine-induced memory impairment. In the present study, multiple memory tasks were included to investigate differences among memory processes (working memory, episodic memory, and metamemory) in the degree to which sedative effects contribute to amnestic effects. Results of a previous interaction study in our laboratory with damphetamine and triazolam at single-dose levels indicated that d-amphetamine significantly reversed triazolams effects on all measures of sedative effects but failed to reverse TRZ-induced impairment on select measures of memory (Mintzer and Griffiths 2003). These results suggest that benzodiazepines do have specific effects on memory that are not merely a by-product of the drugs sedative effects, and that the degree to which sedative effects contribute to the amnestic effects may vary as a function of the particular memory process being assessed. The present study followed up on our previous single-dose study, using a doseeffect design to dissociate the sedative and amnestic effects of triazolam and to test whether there are reliable differences between memory processes in the degree to which sedative effects contribute to the amnestic effects; doses of both triazolam and d-amphetamine were manipulated, enabling the relationship between the amnestic and sedative effects of triazolam to be examined at multiple levels of each effect for each memory process.

Materials and methods Subjects Eighteen adult volunteers (seven women) completed this study. They ranged in age from 18 to 39 years (mean=23) and in weight from 55 to 103 kg (mean=74) and reported having completed 1221 years of education (mean=15). Fourteen participants reported consuming caffeinated beverages delivering 9118 mg of caffeine/day (mean=55), whereas the other three did not report regular consumption of caffeinated beverages. Twelve participants reported

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drinking alcohol socially. One participant reported smoking tobacco cigarettes (seven cigarettes/day). No participants reported histories of drug or alcohol abuse, and all had a negative drug urine screen and breathalyzer test during the initial screening interview. All participants were in good health (as determined by medical history and personal interview) with no contraindications to sedative or stimulant drugs. Individuals with current or past histories of psychiatric disorders were excluded. In the female participants, urine pregnancy tests conducted before the first session were negative. This study was approved by the Institutional Review Board of the Johns Hopkins Bayview Medical Center. Participants gave their written informed consent before beginning the study and were paid for their participation. Participants were requested to refrain from using all psychoactive drugs (with the exception of tobacco and caffeinated products) during the time they were participants in the study. In each session, before drug administration, participants were tested for the presence of various drugs in their urine (benzodiazepines, opioids, methadone, and cocaine) using an EMIT system (Syva, Palo Alto, CA) and the presence of alcohol in expired air using a breathalyzer test. Results of the urinalysis and breathalyzer tests were negative for all participants. General procedures Participants completed a total of nine sessions as outpatients at the Behavioral Pharmacology Research Unit. Participants were informed that during their participation in the study, they would receive various drugs, and that these could include placebo, various sedatives, anxiolytics, stimulants and weight loss medications. Other than receiving this general information, participants were blind to the type of drug administered. Experimental measures (see descriptions below) were administered before drug administration and repeatedly after drug administration (except the episodic memory study and test phases, which were administered at single timepoints after drug administration). The physiological measures and noncomputerized psychomotor measures (i.e., circular lights, balance) were administered approximately 55, 75, 110, 175, 235, and 285 min (285 min, circular lights and balance only) after the first capsule administration timepoint. The participant ratings and DSST were administered approximately 60, 95, 135, 195, and 265 min after the first capsule administration timepoint. The n-back working memory task was administered approximately 140, 200, and 270 min after the first capsule administration timepoint, and the Sternberg working memory task was administered approximately 80, 120, 180, and 250 min after the first capsule administration timepoint. All computerized measures were administered on an Apple Macintosh microcomputer (Apple, Cupertino, CA).

Drug administration Single doses of placebo (PL), triazolam alone (TRZ; 0.25, 0.50 mg/70 kg), d-amphetamine sulfate alone (AMP; 20, 30 mg/70 kg), and TRZ (0.25, 0.50 mg/70 kg) and AMP (20, 30 mg/70 kg) conjointly (at all dose combinations) were administered across nine sessions in a double-blind, withinsubject, crossover design. All drug conditions were administered orally in capsules. To synchronize the peak effects of triazolam and d-amphetamine based on previous pharmacokinetic and behavioral data, a staggered dosing regimen was used in which d-amphetamine was administered 30 min before triazolam. To maintain the double-blind design, during each session, the participant swallowed capsules at two separate timepoints 30 min apart; at each timepoint, the participant received a total of three capsules. Capsules were taken orally with approximately 150 ml of water. The order of drug conditions was determined by two Latin squares using the Williams (1949) method to achieve balance in the presentation order and in the order of drug conditions relative to one another. Triazolam (Halcion; Pharmacia and UpJohn, Kalamazoo, MI) and d-amphetamine (DextroStat; Shire US, Newport, KY) doses were prepared from commercially available 0.25-and 10-mg tablets, respectively. Tablets were crushed, and doses were adjusted by the participants body weight. All doses were dispensed in size 0 capsules. Lactose was used to fill the remainder of the capsules. PL capsules contained only lactose.

Experimental measures Memory tasks Episodic memory/metamemory After an initial study phase, episodic memory (conscious long-term memory for a personally experienced event that is associated with a specific spatial and temporal context; Tulving 1972, 1983) was tested via recognition and free recall tests. Stimuli for each session consisted of a unique set (i.e., no stimulus was repeated across sessions) of 140 common concrete nouns selected from the Thorndike and Lorge (1944) word corpus (i.e., a total of 1,260 words across nine sessions); the sets were equated across the nine sessions for mean word frequency in the language (Thorndike and Lorge 1944) and word length. The 140-word stimulus set assigned to each session was randomly divided into two 70-word subsets with the constraint that the subsets were equated for mean word frequency in the language and word length. In each subset, 35 words represented artificial (i.e., man made) objects, and 35 words represented natural objects. One subset was assigned to the old condition, and one was assigned to the new condition (see below); the subsets

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assigned to the two conditions were counterbalanced such that, across participants, each subset appeared equally often in the old and new condition. During the study phase, which was conducted 120 min after the first capsule administration timepoint (to coincide with the anticipated time of peak effects for both triazolam and d-amphetamine), participants were presented with a set of 70 words (those assigned to the old condition) that appeared on the computer screen one at a time. Each word appeared on the screen for 2 s. To ensure that participants would attend to and encode the words presented during the study phase, participants were asked to perform a conceptual categorization task on each word (to categorize each word as representing an artificial or natural object; e.g., Mintzer et al. 2001) and to make their responses by using the computer mouse to click on the appropriately labeled button on the screen. Two hours after completing the study phase, participants were administered recognition memory and free recall tests. The order of the two tasks was counterbalanced across participants. In the recognition memory test, participants were presented with a set of 140 words that appeared on the screen one at a time in random order; the set included the 70 words that had been presented during the study phase (old) randomly mixed with the 70 words that had not been presented during the study phase (new). Participants were instructed to make judgments about the degree to which they recognized (old) or did not recognize (new) the word from the study phase using a six-point confidence scale (definitely old, probably old, maybe old, maybe new, probably new, and definitely new) and to make their response by clicking on the appropriately labeled button using the computer mouse. Participants were encouraged to distribute their responses across the entire range from 1 through 6. Each test word remained on the screen until the participant responded. In addition to measuring episodic memory, this version of the recognition memory test also provides a measure of the participants metamemory (awareness and knowledge of their own memory; Flavell 1971; Metcalfe and Shimamura 1994) by comparing confidence ratings given to correct vs incorrect recognition responses; presumably, if you are aware of the state of your own memory, you will be more confident in your correct vs incorrect responses. In the free recall test, participants were given 5 min to write down all the words they remembered seeing during the initial study phase. Performance on the conceptual categorization task (study phase) was assessed by analyzing the proportion of correct categorization responses. Two sets of analyses were conducted on the data from the recognition memory test: one in which data were collapsed across confidence ratings (i.e., definitely old, probably old, and maybe old were counted as old responses; definitely new, probably new, and maybe

new were counted as new responses) to provide measures of episodic memory, and one in which the confidence ratings themselves were analyzed to provide a measure of metamemory. Analyses on the collapsed data included the following measures: proportion of old responses made to old words (hit rate), proportion of old responses to new words (false alarm rate), and signal detection measures of sensitivity in distinguishing between old and new words (d) and response bias (C; Snodgrass and Corwin 1988). For both old and new responses, confidence ratings were coded such that maybe=1, probably=2, and definitely=3. Metamemory was measured by calculating the Goodman Kruskal gamma correlation (between confidence ratings and recognition memory accuracy computed for each participant; Goodman and Kruskal 1954). The Goodman Kruskal gamma correlation has been used by other investigators to measure metamemory (cf. Nelson 1984). Values for gamma can range from 1 (complete concordance between confidence ratings and recognition memory accuracy as reflected in high confidence ratings given to correct responses/low confidence ratings given to incorrect responses) to 1 (complete discordance between confidence ratings and recognition memory accuracy). To ensure the availability of the data from a sufficient number of items per participant to enable the performance of these analyses, data were collapsed across old and new words. Dependent measures for the free recall test were number of correct responses (i.e., number of old words; out of a total of 70 possible) and number of (incorrect) intrusions (i.e., number of nonold words) provided by participants.

Working memory Working memory (a type of short-term memory that enables the temporary maintenance and online manipulation of information in the service of behavioral goals; Baddeley 1992) was assessed via the n-back task and a modified Sternberg task. The n-back task (Jonides et al. 1997) assessed the participants ability to recall letters presented n back (0, 1, 2, and 3 back) in a continuous string of letters. For each n back, 60 consonant letters (excluding L, W, and Y) were presented consecutively on the screen for 0.5 s each (interstimulus interval of 3.0 s), and participants were instructed to click the mouse on yes whenever the current letter on the screen matched (target) the letter n positions back in the sequence and to click on no when there was no match (nontarget). For example, in the 2-back task, participants would respond yes to the final M in the sequence mFM; to prevent simple perceptual matching, upper and lower case letters were randomly intermixed. Memory load increases as a function of n. The 0-back is a control condition that involves minimal memory and provides a measure of focused attention only; participants

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were told to click yes whenever the letter on the screen matched a predetermined target letter and to click no whenever there was no match. The probability of a target was approximately 33% in each condition. The order of the four n-back conditions (0, 1, 2, and 3 back) was randomly determined for each participant but was consistent across sessions within a participant. Dependent measures are the proportion of yes responses made to target letters (hit rate), proportion of yes responses made to nontarget letters (false alarm rate), signal detection measures of sensitivity in distinguishing between target and nontarget letters (d) and response bias (C), and median reaction time (RT) on correct trials. In addition, before the start of each n back, participants were asked to rate how well they thought they would perform on the task compared to normal (preperformance estimate). Likewise, after each n back, they were asked to rate how well they thought they had performed compared to normal (postperformance estimate). Participants made these ratings by clicking along a line on the screen labeled much worse at the left extreme and much better at the right extreme, with normal labeled in the middle. Scores range from 50 (much worse) to 50 (much better). These ratings measured the participants cognitive awareness of their performance. The participant estimates of performance were compared to the actual task scores by calculating difference scores as the percentage of the predrug estimate minus the percentage of the predrug actual task score. Thus, a positive score represents an underestimation of performance impairment. The second working memory task was a modified version of the classic Sternberg task (Sternberg 1969) and used procedures similar to those described by Postle et al. (1999) and DEsposito et al. (1999). A memory set consisting of seven randomly selected and randomly ordered consonant letters (excluding L and Y; e.g., PCZMSTF) was presented on the screen followed by a probe consisting of a letter-digit pair (e.g., c-2), and participants were asked to decide whether the probed letter had appeared in the memory set in the ordinal position represented by the digit (e.g., 2=second position in memory set). The probed letter was always a letter that had appeared in the memory set. To prevent simple perceptual matching to the memory set stimuli (which were presented in upper case), the probe was always presented in lower case. Effects on rehearsal processes were tested by varying the delay between memory set presentation and testing (i.e., probe presentation; 0, 12 s). There were 12 trials in each of the two delay conditions and 12 trials in a control condition in which the memory set remained on the screen during probe presentation (i.e., 36 trials total); trials in all conditions were randomly intermixed. The control condition was designed to control for drug effects on nonmemory processes (e.g., motor coordination, percep-

tion, attention, and other cognitive processes). Within each condition, the probed digit represented the correct position of the probed letter on half of the trials. The memory set appeared on the screen for 4 s followed by the predetermined delay and presentation of the probe for a period of 7 s during which the participant responded by using the computer mouse to click on a button labeled yes or no. RT from onset of the probe was recorded. After each response, participants were required to return the mouse cursor to a screen position that was equidistant between the two response buttons; this position was indicated by a circle on the screen that was illuminated when the cursor touched it. There was a 2-s intertrial interval. Dependent measures are the proportion of correct responses and median RT on correct trials. Psychomotor performance tasks Psychomotor performance was measured via circular lights (Griffiths et al. 1983), a standing balance task, and a computerized version of the digit symbol substitution test (DSST; McLeod et al. 1982). Circular lights involved rapid hand-eye coordinated movements in which the participant pressed a series of 16 buttons (circularly arranged around a 54-cm diameter) as rapidly as possible in response to the randomly sequenced illumination of their associated lights. The score was the number of correct button presses during a 60-s trial. The balance task assessed the participants ability to stand upright on one foot with his/her eyes closed and arms extended to the side at shoulder height (for a maximum of 30 s on each foot). For the DSST, in response to randomly selected digits (19) appearing on the screen, participants pressed button positions on a numeric keypad to reproduce the geometric symbol pattern associated with that digit by using the digit-symbol code displayed continuously at the top of the screen. The scores were the number attempted and the proportion correct during a 90-s trial. In addition, awareness of performance was assessed before (preperformance estimate) and after (postperformance estimate) the DSST with the same two questions as described for the n-back task. Participant Ratings Participants rated the strength of drug effect on a five-point scale (coded numerically from 0 to 4), their disliking/liking of the drug effect on a nine-point scale (coded numerically from 4 [maximum disliking] to +4 [maximum liking], with 0 representing neutral), 34 items about their physical and mental state (see Table 1 for specific items) on a fivepoint scale (coded numerically from 0 to 4), and their alertness/sleepiness on a 100-mm visual analog scale

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Table 1 Means and statistical results of comparisons among drug conditions for all measures for which there was a significant main effect of drug condition in the overall ANOVA (ANCOVA for the working memory tasks) Measure PL TRZ 0.25 Participant ratings: sedation Sleepiness (visual analog scale) Sedating/depressant Sleepy Fatigued Tired/lazy Psychomotor performance tasks Balance: s balanced Circular lights: no. of responses DSST: number attempted DSST: proportion correct DSST: estimate (pre) minus actual Working memory tasks N-back: hit rate N-back: false alarm rate N-back: d (sensitivity) N-back: RT (ms) Sternberg: proportion correct Sternberg: RT (ms) Episodic memory tasks Study: proportion correct Free recall: no. correct Recognition: hit rate Recognition: false alarm rate Recognition: d (sensitivity) Gamma (metamemory) Participant ratings: overall Strength of drug effect Drug effect Liking Like effects Good effects Participant ratings: arousal Arousing/stimulant Excited Energetic Participant ratings: other specific Confused Blurred vision Slurred speech Limp Lightheaded/dizzy Unsteady Difficulty concentrating Mentally slow Limbs heavy Forgetful Talkative Comfortable Relaxed Restless/keep moving Shakey/jittery 0.50 AMP 20 10.03 0.32 0.37 0.08 0.15 106.26 104.45 106.69 101.41 2.72 99.73 157.14 101.16 93.64 101.76 98.69 0.97 18.56 0.90 0.22 2.21 0.77 0.93 0.76 0.44 0.71 0.82 0.42 0.23 0.33 0.05 0.06 0.00 0.16 0.25 0.08 0.12 0.22 0.19 0.12 0.37 0.37 0.18 0.16 0.12 30 17.32 0.34 0.89 0.03 0.28 104.50 104.75 108.49 102.06 3.03 100.07 173.12 100.43 95.99 102.58 97.74 0.97 18.89 0.90 0.16 2.56 0.73 1.41 1.05 0.75 0.98 1.01 0.83 0.46 0.74 0.07 0.00 0.03 0.10 0.41 0.21 0.06 0.05 0.18 0.08 0.59 0.22 0.07 0.34 0.41 TRZ/AMP 0.25/20 5.18*c 0.68*c 0.12*c 0.05* 0.08*c 93.48*c 101.31*c 96.70*c 100.19 2.76p 98.67 189.61 96.33 104.44n 101.68* 113.51n 0.96 11.78*p 0.81*c 0.22*c 1.88*c 0.62*c 1.52 1.18 0.95* 1.04* 0.96* 0.51* 0.23 0.26 0.05* 0.22p 0.04 0.29 0.46 0.29 0.24*c 0.20*c 0.46 0.09*c 0.34 0.41 0.66* 0.15 0.20 0.25/30 10.10*c 0.50*c 0.28*c 0.16 0.01*c 86.41*c 101.44*c 96.48*c 100.42 2.62p 99.66 167.39 97.93 107.71n 101.90* 107.22p 0.96 11.67*p 0.81*c 0.23p 1.83*c 0.61*c 1.56 1.33 1.32* 1.39* 1.30* 0.78* 0.62* 0.61* 0.16 0.05*c 0.02 0.22 0.54 0.28 0.31*c 0.24*c 0.51 0.08*c 0.88* 0.44 0.46 0.42* 0.25* 0.50/20 1.31*c 0.87*p 0.24*c 0.26 0.11*c 58.93n 91.02*c 89.65*p 98.51*c 9.51n 91.82n 196.01*c 86.32n 118.89*n 94.65p 125.02n 0.94n 6.33n 0.75n 0.33n 1.32*p 0.42n 1.87 1.55 0.80* 1.18* 1.12* 0.70* 0.47 0.36* 0.19*c 0.27n 0.02 0.50n 0.78n 0.55n 0.61*p 0.43*c 0.35p 0.21p 0.67* 0.23 0.29 0.33* 0.27* 0.50/30 2.94*c 0.83*p 0.56*c 0.14 0.19*c 60.54n 93.85*c 88.78*p 97.14*c 11.53n 95.57*p 298.31n 90.01*p 119.24*n 95.70*c 116.03n 0.92n 6.28n 0.68n 0.32n 1.09n 0.34n 1.97 1.60 1.04* 1.24* 1.20* 0.69* 0.67* 0.46* 0.21*c 0.33n 0.19 0.33n 0.77n 0.60n 0.60*p 0.53*c 0.46n 0.15*c 0.81* 0.24 0.61* 0.37* 0.23

1.17 0.35 0.14 0.21 0.08 98.39 97.63 101.75 100.33 3.28 102.04 196.84 101.11 96.31 99.73 103.83 0.97 16.89 0.85 0.19 2.12 0.71 0.46 0.43 0.00 0.15 0.14 0.05 0.00 0.06 0.11 0.02 0.00 0.02 0.14 0.10 0.11 0.19 0.11 0.09 0.06 0.09 0.07 0.06 0.00

19.47 1.19 0.93 0.39 0.72 70.16 89.84 87.59 96.95 7.99 97.63 161.48 99.18 105.58 94.52 114.62 0.95 7.56 0.72 0.29 1.26 0.44 1.70 1.30 0.21 0.37 0.47 0.02 0.04 0.16 0.27 0.40 0.09 0.21 0.35 0.26 0.79 0.67 0.33 0.42 0.02 0.08 0.10 0.06 0.00

23.97 1.85 1.37 0.42 0.84 45.50 75.91 78.77 91.40 14.06 88.18 309.11 79.58 111.51 89.27 126.17 0.92 4.24 0.67 0.39 0.87 0.38 2.28 1.82 0.18 0.62 0.61 0.14 0.19 0.14 0.48 0.48 0.10 0.50 0.65 0.71 1.21 1.04 0.66 0.47 0.17 0.07 0.11 0.06 0.00

Psychopharmacology (2007) 192:425440 Table 1 (continued) Measure PL TRZ 0.25 0.96 0.98 2.39 AMP 20 TRZ/AMP 0.25/20

431

0.50

30

0.25/30

0.50/20

0.50/30

Physiological Systolic blood pressure Diastolic blood pressure Heart rate

1.17 1.55 5.05

0.15 0.10 2.05

11.67 7.72 0.94

19.81 13.22 0.39

15.38* 4.68* 1.18

18.68* 11.72* 0.40

14.79* 9.87* 4.98

19.54* 13.90* 6.40

For participant ratings and physiological measures, the analyzed data were difference scores from predrug values. For working memory and psychomotor performance tasks, the analyzed data were percentages of predrug values. AUC values are shown for all measures except the episodic memory tasks, which were administered at a single timepoint. Bold indicates a significant difference between active drug and PL. An asterisk (*) indicates a significant difference between TRZ/AMP and the corresponding TRZ dose. For TRZ/AMP combination doses, c indicates complete reversal by AMP of TRZs effects, p indicates partial reversal, n indicates no reversal (see text for definitions), and the absence of c, p, or n indicates that the combination dose was excluded from categorization for that measure (if the corresponding TRZ vs PL comparison was not significant, the combination doses were excluded from categorization; measures with an AMP vs PL comparison in the same direction as the significant TRZ vs PL comparison were also excluded from categorization; see text for details).

(labeled very alert and sleepy at the left and right extremes, respectively). Physiological measures Blood pressure (systolic and diastolic) and heart rate were measured using the Sentry II system (NBS Medical, Costa Mesa, CA). Respiration rate was measured manually (by observation over a 15-s duration). Data analysis Data from two participants were missing for several measures in the TRZ 0.50 condition at two timepoints because of strong drug effects that prevented them from completing assessments at those timepoints, and data from one participant were missing for the DSST in the AMP 30 condition at one timepoint because of a technical problem. Gamma correlations could not be computed for two participants in at least one drug condition because they failed to use the entire confidence rating scale. Therefore, Proc mixed (a procedure in SAS that models missing data) was used for all analyses. For participant ratings and physiological measures, the analyzed data were difference scores from predrug values. For working memory and psychomotor performance tasks, the analyzed data were percentages of predrug values. Area under the timecourse curve (AUC) values were calculated for each participant by the method of trapezoids. Standard analyses Data were analyzed by repeated measures analysis of variance (ANOVA) with drug condition (PL; TRZ 0.25, 0.50; AMP 20, 30; and TRZ/AMP 0.25/20, 0.25/30, 0.50/20, and 0.50/30) and time (see above for specific timepoints) as factors. A second set of analyses was conducted using repeated measures one factor ANOVA with drug condition as the factor; data analyzed were data

from the episodic memory/metamemory tasks administered only at a single timepoint and AUC data. Data from the working memory tasks were analyzed by analysis of covariance (ANCOVA) with the corresponding nonmemory control condition as covariate and drug condition and memory load (1, 2, and 3 back; n-back task) or delay (0, 12 s; Sternberg task) as factors. Data from the free recall and recognition memory tests initially were analyzed with the order of the two tasks (which was counterbalanced across participants; see Materials and methods) as a between subjects factor. However, given that there were no significant interactions between drug condition and task order, analyses in which data were collapsed across the two task orders are reported. For all statistical tests, p0.05 was considered significant. Significant main effects and interactions were followed up with simple effects tests as appropriate and modified Bonferroni corrections were used (cf. Keppel 1991). Given that the focus of the study was AMPs reversal of TRZs effects, comparisons among doses were limited to the following: 1. TRZ vs PL (i.e., TRZ 0.25 vs PL; TRZ 0.50 vs PL) 2. AMP vs PL (i.e., AMP 20 vs PL; AMP 30 vs PL) 3. TRZ/AMP vs TRZ (i.e., TRZ/AMP 0.25/20 vs TRZ 0.25; TRZ/AMP 0.25/30 vs TRZ 0.25; TRZ/AMP 0.50/ 20 vs TRZ 0.50; TRZ/AMP 0.50/30 vs TRZ 0.50) Measures for which the TRZ vs PL comparison was significant were categorized in terms of the degree of reversal of the TRZ effect (complete, partial, and none) in each of the relevant TRZ/AMP combination conditions using the following definitions (measures with an AMP vs PL comparison in the same direction as the significant TRZ vs PL comparison were excluded from categorization): 1. Complete=both of the following are true: a. TRZ/AMP vs TRZ was significant b. TRZ/AMP vs PL was not significant

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2. Partial=only one of the following is true: a. TRZ/AMP vs TRZ was significant b. TRZ/AMP vs PL was not significant 3. None=both of the following are true: a. TRZ/AMP vs TRZ was not significant b. TRZ/AMP vs PL was significant

z-Score standardized analyses To facilitate the comparison of AMPs reversal of TRZs effects across measures, an additional set of analyses was conducted on data from the sedative and memory measures (see Table 2 for specific measures). For purposes of this analysis, sedative measures included participant ratings of sedative effects and psychomotor performance tasks. To limit the number of measures, a single measure was selected for assessments with multiple dependent variables (e.g., for the DSST, only number of trials attempted was included). The analyzed data for the assessments administered repeatedly were AUC. Analyzed data for the working memory tasks were the control-task adjusted values from the ANCOVA collapsed across memory load and delay as appropriate. Data from these measures were first converted to z scores for each participant. Next, difference scores were

computed for each participant for each measure between the PL value for that measure and the value of each of the following doses for that measure: TRZ 0.25, 0.50 (indices of the TRZ effect for that measure) and TRZ/AMP 0.25/20, 0.25/30, 0.50/20, 0.50/30 (indices of the combination effect for that measure). To ensure that the direction of the effect was consistent across measures, difference scores for measures in which a TRZ effect is indicated by a higher value for TRZ vs PL (e.g., RT measures; participant ratings of sedative effects), were computed by subtracting the PL value from the active drug value, whereas difference scores for measures in which a TRZ effect is indicated by a lower value for TRZ vs PL (e.g., accuracy measures) were computed by subtracting the active drug value from the PL value. The following sets of planned comparisons were conducted on these difference scores as follows: 1. TRZ/AMP vs TRZ for each measure (i.e., TRZ/AMP 0.25/20 vs TRZ 0.25; TRZ/AMP 0.25/30 vs TRZ 0.25; TRZ/AMP 0.50/20 vs TRZ 0.50; TRZ/AMP 0.50/30 vs TRZ 0.50). A significant difference suggests significant reversal by AMP of the TRZ effect for that measure. 2. Comparisons between pairs of specific memory measures at each of the six doses. A significant difference between measures at the combination doses suggests differential reversal by AMP of the TRZ effect

Table 2 Mean difference scores for TRZ minus PL (columns 23) and TRZ/AMP minus PL (columns 47) calculated from z-score standardized data for the sedative and memory measures and results of planned comparisons conducted on these data Measure TRZ minus PL 0.25 Sedative Participant ratings: sedation Sleepiness (visual analog scale) Sedating/depressant Sleepy Tired/lazy Psychomotor performance tasks Balance: s balanced Circular lights: no. responses DSST: no. attempted Memory Working memory tasks N-back: d (sensitivity) N-back: RT (ms) Sternberg: proportion correct Sternberg: RT (ms) Episodic memory tasks Free recall: no. correct Recognition: d (sensitivity) Gamma (metamemory) 0.50 TRZ/AMP minus PL 0.25/20 0.25/30 0.50/20 0.50/30

0.82 0.96 0.88 0.73 0.91 0.58 1.15

1.06 1.70 1.29 0.90 1.74 1.62 1.91

0.28 0.38 0.01 0.00 0.16 0.27 0.41

0.50 0.17 0.11 0.10 0.39 0.28 0.43

0.11 0.59 0.09 0.04 1.28 0.49 0.98

0.18 0.55 0.35 0.12 1.22 0.28 1.05

0.53a 0.90a 0.62a 0.87a 1.07a 0.95a 0.87a

1.84a 1.52ab 1.54ab 1.77a 1.50ab 1.38ab 1.05b

0.40a 0.69a 0.32b 0.50a 0.59a 0.27ab 0.29ab

0.29a 0.73a 0.33b 0.22ab 0.60a 0.32a 0.33a

1.15a 1.79c 0.49b 1.34ac 1.21ac 0.89ab 0.90ab

0.91ab 1.93c 0.47b 0.99ab 1.22a 1.13a 0.94ab

Bold indicates a significant difference between TRZ/AMP and the corresponding TRZ dose (i.e., significant reversal by AMP of the TRZ effect for that measure). In the memory section of the table, letters a, b, and c are used to indicate comparisons between pairs of the seven memory measures at each dose; within the same column, any two means designated with the same letter are not significantly different from each other.

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(assuming there is no significant difference between these measures at the corresponding TRZ alone dose). 3. Comparisons between memory measures (collapsed across all memory measures) and sedative measures (collapsed across all sedative measures) at each of the six doses. These comparisons test the hypothesis that the combination effect is significantly greater (i.e., less reversal) for memory vs sedative measures (whereas no difference is hypothesized between the measures for the TRZ effect).

Results Timecourse of effects Before presenting the dose effect data, timecourse data are briefly summarized here, and representative timecourse functions are shown in Fig. 1. As expected, both TRZ and AMP produced orderly time-related increases relative to PL in participant ratings of overall drug effect (e.g., strength of

Fig. 1 Timecourse functions (TRZ and AMP, alone and in combination) for participant rating of strength of drug effect, performance on the balance task, and systolic blood pressure. x-Axis, time in minutes after the first capsule administration timepoint; d-amphetamine was administered at the first capsule administration timepoint, and triazolam was administered 30 min later; 0 indicates predrug. Unadjusted means + 1 S.E.M are shown (excluding missing data). Filled symbols indicate active drug values that are significantly different from the corresponding PL value at the same timepoint

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drug effect). TRZ also produced time-related decrements in psychomotor performance (e.g., balance) and AMP produced increases in physiological measures (e.g., systolic blood pressure). Given that various assessments were administered at different times, it is difficult to determine the exact time of peak drug effects; however, for both TRZ and AMP, effects appeared to peak somewhere between approximately 75 and 140 min after the first capsule administration timepoint. Dose effects Results across all drug conditions are shown in Table 1. Effects of TRZ alone TRZ produced significant dose-related increases relative to PL in participant ratings of sedative effects and significant decrements in psychomotor performance (Fig. 2). TRZ also significantly impaired DSST performance estimates (preperformance only) such that before performing the DSST, participants underestimated their degree of impairment. The ANCOVAs on the working memory tasks revealed no significant interaction between dose and memory load (n-back) or delay (Sternberg). Therefore, data are shown in Table 1 and Fig. 3 collapsed across memory load and delay. As expected, TRZ produced significant dose-related decrements in working memory accuracy (TRZ 0.50 only) and increases in RT on both tasks. During the study phase of the episodic memory task, TRZ (0.50 only) significantly decreased the proportion of correct categorization responses relative to PL. On the free recall test, TRZ produced a significant dose-related decrease in the number of correct responses (Fig. 4) but did not significantly affect the number of intrusion responses relative to PL (data not shown). On the recognition memory test, TRZ produced significant dose-related decreases in hit rate and d (Fig. 4) and a significant increase in false alarm rate relative to PL; TRZ did not affect the response bias measure C (data not shown). TRZ also produced a significant dose-related decrease in the Gamma correlation, indicating impaired metamemory. TRZ also produced significant dose-related increases in participant ratings of overall drug effects and a number of specific items. TRZ produced a significant increase in heart rate relative to PL (TRZ 0.50 only) but did not significantly affect blood pressure or respiration rate. Effects of AMP alone AMP (30 only) produced significant increases in all participant ratings of arousal and significant decreases in some participant ratings of sedative effects relative to PL (Fig. 2). AMP also produced significant doserelated increases in participant ratings of overall drug effects (including liking and good effects) and of a number of specific items. AMP (30 only) significantly enhanced

Fig. 2 AUC doseeffect functions (TRZ, AMP, and TRZ/AMP) for participant rating of sleepiness (visual analog scale) and performance on the circular lights task. Unadjusted means 1 S.E.M are shown (excluding missing data). Filled symbols indicate active drug values that are significantly different from PL; an asterisk indicates a significant difference between TRZ/AMP and the corresponding TRZ dose

performance on the DSST (number attempted) and circular lights relative to PL but did not significantly affect balance performance. AMP did not significantly enhance working memory, performance during the study phase of the episodic memory task, free recall, or metamemory but did produce a significant increase in d on the recognition memory test (AMP 30 only). AMP produced significant dose-related increases relative to PL in both systolic and diastolic blood pressure but did not significantly affect heart rate or respiration.

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435

Fig. 3 AUC doseeffect functions (TRZ, AMP, and TRZ/AMP) for measures of performance on the working memory tasks (n-back and Sternberg). Control-task adjusted means from the ANCOVA collapsed

across memory load (n-back) and delay (Sternberg) are shown. Symbols are identical to those described for Fig. 2

Reversal by AMP of TRZs effects AMP produced complete reversal (as defined above) of TRZs effects at all four dose combinations on all participant ratings of sedative effects (Fig. 2) except sedating/depressant that showed complete reversal at 0.25/20 and 0.25/30 but only partial reversal at 0.50/20 and 0.50/30. AMP produced complete reversal at all four combinations for circular lights (Fig. 2) but only at 0.25/20 and 0.25/30 for balance. For the DSST number of trials attempted, AMP produced complete reversal at 0.25/ 20 and 0.25/30 but only partial reversal at 0.50/20 and 0.50/ 30. For DSST proportion correct, AMP produced complete reversal at 0.50/20 and 0.50/30 (the only relevant combinations because TRZ 0.25 was not significantly different from PL). For DSST preperformance estimates, there was only partial reversal at 0.25/20 and 0.25/30 and no reversal at 0.50/20 and 0.50/30. Participant ratings of other specific

effects varied in the degree of reversal of TRZs effects by AMP, with ratings of mental state (confused, difficulty concentrating, mentally slow, and forgetful) tending to produce more reversal than ratings of somatic state (blurred vision, limp, lightheaded/dizzy, unsteady, and limbs heavy). AMP did not produce complete reversal of TRZs effects at all four dose combinations on any of the memory measures. The memory measures ranged in degree of reversal from none across all relevant combinations (i.e., combinations for which the corresponding TRZ dose was significantly different from PL; n-back working memory task, RT) to none across none of the relevant combinations (Sternberg working memory, proportion correct; only 0.50/ 20 and 0.50/30 relevant because TRZ 0.25 was not significantly different from PL) with most of the memory measures showing an overall pattern of partial reversal

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Comparisons between pairs of memory measures at each of the four combinations (see the memory section of Table 2) revealed that the combination effect was significantly greater (i.e., less reversal) for RT on the n-back working memory task relative to all other measures for one or both of the 0.50 combinations (i.e. 0.50/20, 0.50/30), and that the combination effect was significantly smaller (i.e., more reversal) for proportion correct on the Sternberg working memory task relative to all other measures for at least one of the four combinations. Results of the comparisons between memory measures (collapsed across all seven memory measures in Table 2) and sedative measures (collapsed across all seven sedative measures in Table 2) at each of the six doses are shown in Fig. 5. As predicted, the combination effect was significantly greater (i.e., less reversal) for memory vs sedative measures at each of the four combinations, whereas there was no significant difference between the measures for the TRZ alone effect at either TRZ dose.

Discussion Effects of triazolam and d-amphetamine alone The observed effects of triazolam alone (increases in participant ratings of sedative effects, impaired psychomotor performance, working memory, episodic memory, and metamemory) replicate results of previous studies with triazolam and other benzodiazepines (for reviews, see Curran 1991, 2000; Hollister et al. 1993; Woods et al. 1992). Likewise, effects of d-amphetamine alone (increases in participant ratings of arousal and blood pressure, enhanced psychomotor performance) replicate previous findings with amphetamine (for reviews, see Solanto 1998; Weiss and Laties 1962) including those of our previous interaction study (Mintzer and Griffiths 2003). Consistent with previous demonstrations that amphetamine enhances the speed but not the accuracy of psychomotor performance (Fleming et al. 1995; Mintzer and Griffiths 2003; Rapoport et al. 1980; Robbins and Everitt 1982), damphetamines enhancement of DSST performance was reflected in an increase in the number of digits attempted but not in the proportion of digits correct (Table 1). Previous studies examining amphetamines acute effects on memory in healthy adults do not yield a consistent pattern of results. In early studies focused on the acquisition of stimulusresponse relationships, there are reports of enhancement by amphetamine (i.e., fewer trials to learning criterion; Weitzner 1965), no effect (Kornetsky 1958; Hurst et al. 1969), and even impairment (Burns et al. 1967). Although results of one study suggest no effect on shortterm/working memory (Soetens et al. 1995), results of

Fig. 4 Doseeffect functions for measures of performance on the free recall and recognition memory tests. Unadjusted means 1 S.E.M are shown (excluding missing data). Symbols are identical to those described for Fig. 2

(Figs. 3 and 4). It should be noted that for the n-back working memory task, RT was actually significantly longer (i.e., worse performance) for TRZ/AMP 0.50/20 and 0.50/ 30 relative to TRZ 0.50. Results of the planned comparisons conducted on the difference scores calculated for each participant from zscore standardized data for the sedative and memory measures are shown in Table 2. The TRZ/AMP vs TRZ comparisons for each measure revealed an overall pattern of reversal similar to that of the standard analyses described above. Thus, participant ratings of sedative effects and circular lights showed significant reversal (i.e., a significant TRZ/AMP vs TRZ difference) at all four combinations (except sedating/depressant that showed significant reversal at all but the 0.25/20 combination), whereas balance showed significant reversal only at 0.25/20. Again, the memory measures ranged in degree of reversal from no significant reversal at any combination (n-back working memory task, RT; free recall, number of correct responses; gamma correlation) to significant reversal at all four combinations (Sternberg working memory, proportion correct).

Psychopharmacology (2007) 192:425440 Fig. 5 Difference scores (TRZ minus PL; TRZ/AMP minus PL) calculated from z-score standardized data for the sedative measures (collapsed across all seven sedative measures in Table 2) and memory measures (collapsed across all seven memory measures in Table 2). Unadjusted means + 1 S.E.M are shown (excluding missing data). An asterisk indicates a significant difference between the sedative and memory variables in that condition

437

another study (Kennedy et al. 1990) suggest enhancement. Results of recent neuroimaging and genetic studies suggest that individual differences in amphetamines effects on working memory may be related to differences in dopamine function, which has been shown to vary across genotypes (Mattay et al. 2000, 2003). Specifically, an inverted Ushaped function seems to characterize the relationship between dopamine function and working memory performance such that performance is optimal at moderate levels of dopamine and worse at lower and higher dopamine levels. Thus, working memory is enhanced (as evidenced by enhanced performance on the n-back task and/or enhanced efficiency of relevant prefrontal cortical networks) after acute d-amphetamine administration in individuals with lower baseline prefrontal dopamine function and unchanged or reduced in individuals with higher baseline prefrontal dopamine function. In the present study, d-amphetamine did not enhance working memory performance on either the n-back or Sternberg task. Although the lack of enhancement of the accuracy measures may be an artifact of the near-ceiling accuracy on both tasks in the placebo condition, the lack of enhancement of RT is unlikely due to ceiling effects. To test the hypothesis that the degree of enhancement by d-amphetamine of working memory performance is inversely related to baseline level of working memory performance as suggested by previous studies, we performed secondary analyses on data from the n-back (d; control task-adjusted values, collapsed across

memory load) and Sternberg (proportion correct; controltask adjusted values, collapsed across delay) working memory tasks. Specifically, for each measure, we calculated Pearson correlations between the value at the predrug timepoint (collapsed across all nine drug conditions) and an enhancement score (calculated for each participant as the AUC value for d-amphetamine minus the AUC value for placebo, divided by the AUC value for placebo as a baseline); Bonferroni corrections were used. For the n-back task (similar to the task used by Mattay et al. 2000), there was a significant negative correlation between baseline performance and the d-amphetamine enhancement score for the 20-mg d-amphetamine dose (r=0.67, p=0.002) as predicted. The correlation was not significant for the 30-mg d-amphetamine dose. There were no significant correlations for the Sternberg task. Few studies have examined amphetamines effects on episodic memory. Rapoport et al. (1980) reported enhancement of free recall but not cued recall performance, whereas Hurst et al. (1969) reported enhancement of cued recall performance. Soetens et al. (1995) reported enhancement of both free recall and recognition memory performance; however, the timing of stimulus presentation relative to drug administration was designed to measure effects on consolidation processes rather than on encoding or retrieval. In the present study, d-amphetamine did not affect free recall or metamemory but significantly enhanced d on the recognition memory test (only at the 30-mg dose). The

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study phase of the episodic memory task was timed to coincide with the anticipated time of peak effects for both triazolam and amphetamine to measure effects on encoding. Inconsistencies in effects of amphetamine on memory across studies may be related to individual differences in response to amphetamine. In addition to the effects of differences in dopamine function discussed above, there is evidence that the response to amphetamine varies as a function of gender and hormone levels (Justice and de Wit 2000a,b; White et al. 2002) although the effects of these variables have not been tested on memory specifically. Consistent with the results of our previous interaction study (Mintzer and Griffiths 2003), d-amphetamine did not affect metamemory in the present study. To our knowledge, other studies have not examined effects of amphetamine on metamemory. Reversal by d-amphetamine of triazolams effects In the standard analyses, d-amphetamine produced a complete reversal of triazolams effects at all four dose combinations on all but one participant rating of sedative effects and complete or partial reversal of triazolams effects on all psychomotor performance measures except the balance task. The planned comparisons conducted on zscore standardized data revealed a similar pattern of results for the sedative measures and support the overall conclusion that the sedative effects of triazolam were reversed by d-amphetamine. The finding of complete or partial reversal for ratings of specific aspects of mental state associated with sedation (confused, difficulty concentrating, mentally slow, and forgetful) is also consistent with this conclusion. Interestingly, ratings of somatic state (blurred vision, limp, lightheaded/dizzy, unsteady, and limbs heavy) showed less reversal than ratings of mental state. This pattern is consistent with the finding of less reversal for the balance task (a measure of basic motor coordination) and suggests that the physical aspects of sedation may be dissociated from the mental aspects. As discussed in detail below, the memory measures varied in the degree of reversal by d-amphetamine of triazolams effects. However, it is important to note that, overall, the memory measures exhibited a pattern of less reversal than the sedative measures. This conclusion is supported by both the standard and z-score standardized analyses. Specifically, in the standard analyses, d-amphetamine did not produce complete reversal of triazolams effects at all four dose combinations on any of the memory measures, and in the z-score standardized analyses, the combination effect was significantly greater (i.e., less reversal) for memory vs sedative measures at each of the four combinations (whereas there was no significant difference between the measures for the triazolam alone

effect at either triazolam dose; Fig. 5). Thus, results of the present doseresponse study are consistent with those of our previous single-dose interaction study (Mintzer and Griffiths 2003) and provide further evidence that the amnestic and sedative effects of benzodiazepines can be dissociated. In the standard analyses, the memory measures ranged in degree of reversal from none across all relevant combinations (n-back working memory task, RT) to none across none of the relevant combinations (Sternberg working memory task, proportion correct) with most of the memory measures showing an overall pattern of partial reversal (Table 1; Figs. 3 and 4). In general, there was relatively more reversal by d-amphetamine at the 0.25-mg triazolam dose than at the 0.50-mg dose, but there were differences among relevant measures at the 0.25-mg dose such that some measures showed complete reversal (recognition, hit rate, d; gamma correlation), whereas others showed partial reversal (Sternberg, RT; free recall, number correct; recognition, false alarm rate) or none (n-back, RT). Thus, the doseeffect interaction design enables differences to emerge between measures in the relative contribution of triazolam-induced sedative effects to triazolam-induced memory effects. The present study was designed to focus on the relationship between the sedative and amnestic effects of benzodiazepines. However, it is important to note that in addition to sedation, benzodiazepines produce other effects that may also contribute to their memory-impairing effects. For example, benzodiazepines have been shown to impair focused and selective attention (for a review, see BuffettJerrott and Stewart 2002). The nonmemory control conditions used in the two working memory tasks were designed to control for effects on attention. However, there were no analogous controls for the episodic memory tasks. Thus, it is possible that benzodiazepine-induced effects on attention may have contributed to the observed memory impairment in these tasks. Given that sedation may also play a role in attentional effects, it is also possible that the contribution of sedation to memory impairment was actually mediated by attentional mechanisms. Further adding to the complexity, d-amphetamine has been shown to enhance attention (for reviews, see Solanto 1998; Weiss and Laties 1962). The present study was not designed to disentangle the complex interrelationships among sedation, attention, memory, and other processes. However, these issues are intriguing and warrant exploration in future studies. From a methodological perspective, the z-score standardized analysis technique is interesting and enables effects (both the triazolam effect and the triazolam/ amphetamine combination effect in this study) to be directly compared statistically across different measures using the same metric. For example, direct comparison of the combination effect for the collapsed sedative measures

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439 benzodiazepine antagonist, flumazenil. Psychopharmacology 103:519523 Curran HV, Bond A, OSullivan G, Bruce M, Marks I, Lelliot P, Shine P, Lader M (1994) Memory functions, alprazolam and exposure therapy: a controlled longitudinal study of patients with agoraphobia and panic disorder. Psychol Med 24:969976 Curran HV, Pooviboonsuk P, Dalton JA, Lader MH (1998) Differentiating the effects of centrally acting drugs on arousal and memory: an event-related potential study of scopolamine, lorazepam and diphenhydramine. Psychopharmacology 135:2736 DEsposito M, Postle BR, Ballard D, Lease J (1999) Maintenance versus manipulation of information held in working memory: an event-related fMRI study. Brain Cogn 41:6686 Flavell JH (1971) What is memory development the development of ? Hum Dev 14:272278 Fleming K, Bigelow LB, Weinberger DR, Goldberg TE (1995) Neuropsychological effects of amphetamine may correlate with personality characteristics. Psychopharmacol Bull 31:357362 Ghoneim MM (2004a) Drugs and human memory (part 1): clinical, theoretical, and methodologic issues. Anesthesiology 100:9871002 Ghoneim MM (2004b) Drugs and human memory (part 2): clinical, theoretical, and methodologic issues. Anesthesiology 100:12771297 Ghoneim MM, Mewaldt SP, Berie JL, Hinrichs V (1981) Memory and performance effects of single and 3 week administration of diazepam. Psychopharmacology 73:147151 Goodman LA, Kruskal WH (1954) Measures of association for crossclassifications. J Am Stat Assoc 49:732764 Green JF, McElholm A, King DJ (1996) A comparison of the sedative and amnestic effects of chlorpromazine and lorazepam. Psychopharmacology 128:6773 Griffiths RR, Bigelow GE, Liebson I (1983) Differential effects of diazepam and pentobarbital on mood and behavior. Arch Gen Psychiatry 40:865873 Hollister LE, Mller-Oerlinghausen B, Rickels K, Shader RI (1993) Clinical uses of benzodiazepines. J Clin Psychopharmacol 13:1S169S Hommer D, Weingartner H, Breier A (1993) Dissociation of benzodiazepine-induced amnesia from sedation by flumazenil. Psychopharmacology 112:455460 Hurst PM, Radlow R, Chubb NC, Bagley SK (1969) Effects of Damphetamine on acquisition, persistence, and recall. Am J Psychol 82:307319 Jonides J, Schumacher EH, Smith EE, Lauber EJ, Awh E, Minoshima S, Koeppe RA (1997) Verbal working memory load affects regional brain activation as measured by PET. J Cogn Neurosci 9:462475 Justice AJH, de Wit H (2000a) Acute effects of d-amphetamine during the early and late follicular phases of the menstrual cycle in women. Pharmacol Biochem Behav 66:509515 Justice AJH, de Wit H (2000b) Acute effects of estradiol pretreatment on the response to d-amphetamine in women. Neuroendocrinology 71:5159 Kennedy RS, Odenheimer RC, Baltzley DR, Dunlap WP, Wood CD (1990) Differential effects of scopolamine and amphetamine on microcomputer-based performance tests. Aviat Space Environ Med 61:615621 Keppel G (1991) Design and analysis: a researchs handbook, 3rd edn. Prentice Hall, Englewood Cliffs Kirk T, Roache JD, Griffiths RR (1990) Doseresponse evaluation of the amnestic effects of triazolam and pentobarbital in normal subjects. J Clin Psychopharmacol 10:160167 Kornetsky C (1958) Effects of meprobamate, phenobarbital and dextroamphetamine on reaction time and learning in man. J Pharmacol Exp Ther 123:216219 Mattay VS, Callicott JH, Bertolino A, Heaton I, Frank JA, Coppola R, Berman KF, Goldberg TE, Weinberger DR (2000) Effects of

vs collapsed memory measures (shown in Fig. 5) enabled the sedative and memory measures to be dissociated statistically, thus strengthening the conclusion that the sedative and amnestic effects of benzodiazepines can be dissociated. The z-score standardized analysis technique also enabled statistical comparisons to be made between pairs of memory measures. The conclusions of relatively less reversal for RT on the n-back working memory task and relatively more reversal for proportion correct on the Sternberg working memory task were supported both by the standard analyses and by the comparisons conducted on the z-score standardized data and appear to be reliable. Given that we did not specifically predict these differences between memory measures, we will not speculate on possible underlying mechanisms. However, these differences are intriguing and warrant exploration in future studies. In summary, using both standard analyses and a novel zscore standardized analysis technique, this doseeffect interaction study provides strong evidence that benzodiazepines have specific effects on memory that are not merely a by-product of the drugs sedative effects, and that the degree to which sedative effects contribute to the amnestic effects varies as a function of the particular memory process being assessed.
Acknowledgment This project was supported by the National Institute on Drug Abuse Research Grant DA-11936. The authors thank Crystal Barnhouser, Kristina Burns, and Koty Nadeau for protocol management, John Yingling for computer programming assistance and technical support, and Linda Felch and Paul Nuzzo for assistance with data analysis.

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