Apomorphine effects on episodic memory in young healthy volunteers

Neuropsychologia 46 (2008) 292–300

Apomorphine effects on episodic memory in young healthy volunteers Alonso Montoya, Samarthji Lal, Matthew Menear, Elisabeth Duplessis, Joseph Thavundayil, Norbert Schmitz, Martin Lepage ∗ Douglas Mental Health University Institute, Department of Psychiatry, McGill University, Canada Received 21 December 2006; received in revised form 13 July 2007; accepted 16 July 2007 Available online 26 July 2007

Abstract Rationale: Dopamine (DA) modulates working memory. However, the relation between DA systems and episodic (declarative) memory is less established. Frontal lobe DA function may be involved. We were interested in assessing whether apomorphine (Apo), a drug used extensively in clinical research as a probe of DA function, has an effect on episodic memory test performance in healthy volunteers. Objective: To investigate the effect of a presynaptic dose of Apo on episodic memory tests and on other tests thought to be sensitive to frontal lobe functions. Methods: Twenty healthy subjects were treated with Apo HCl (5 ␮g/kg sc) or placebo (10 subjects/group) in a randomized, double blind parallel group design and performance on a battery of cognitive tests was assessed. Results: Apomorphine significantly impaired performance on tests of source recognition (d.f. = 19, p = 0.05) and item recognition memory (d.f. = 19, p < 0.05), and memory interference (d.f. = 19, p < 0.010). No significant change was found on other tests (Go/no-Go Test, Categorized Words, Stroop, Trail Making Test, and verbal fluency). Conclusion: Findings in this small sample of subjects suggest that dopaminergic transmission affects episodic memory functions. © 2007 Elsevier Ltd. All rights reserved. Keywords: Episodic memory; Declarative memory; Executive functions; Dopamine; Apomorphine; Prefrontal cortex; Hippocampus; Recall; Recognition

1. Introduction Pharmacological studies in both humans and animals have established a link between working memory functions and dopamine (DA) activity in the brain (Brozoski, Brown, Rosvold, & Goldman, 1979; Fournet, Moreaud, Roulin, Naegele, & Pellat, 2000; Robbins, 2000). Working memory refers to a memory system involved in the temporary maintenance and manipulation of information for behavioral purposes (Baddeley, 1992). In healthy humans, the administration of DA receptor agonists improves working memory abilities (Luciana & Collins, 1997; Mehta, Swainson, Ogilvie, Sahakian, & Robbins, 2001; Muller, von Cramon, & Pollmann, 1998), whereas, the admin-

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Corresponding author at: Douglas Mental Health University Institute, FBC, 6875 boul. LaSalle, Verdun, Quebec H4H 1R3, Canada. E-mail address: [email protected] (M. Lepage). 0028-3932/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropsychologia.2007.07.012

istration of DA receptor antagonists impairs working memory (Luciana & Collins, 1997; Mehta, Sahakian, McKenna, & Robbins, 1999). The association between DA activity and other memory systems is less clear. Little is known for instance about the role of DA in episodic (declarative) memory, the memory system responsible for the explicit and conscious recollection of events (Tulving, 1983). There is reason to believe that there may be a relationship as many dopamine receptors are found in areas of the brain known to be implicated in episodic memory. For instance, DA receptors are found in the prefrontal cortex (Grace, 2002), subcortical regions (caudate, putamen, thalamus, amygdala) (Gurevich & Joyce, 1999; Schatzberg & Nemeroff, 1995), as well as hippocampal regions (Ryoo & Joyce, 1994). Numerous lesion and neuroimaging studies have shown these same regions to be involved in episodic memory processing (Exner, Weniger, & Irle, 2001; Lepage, Ghaffar, Nyberg, & Tulving, 2000; Rugg, Fletcher, Chua, & Dolan, 1999; Tulving, 2002). Thus, a closer exam-

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ination of the role of DA in episodic memory is clearly warranted. To date, only a few studies in humans have explored the effect of DAergic agents on episodic memory performance. l-Dopa improves word learning in normal humans (Knecht et al., 2004). However, l-dopa, a precursor of both dopamine and norepinephrine increases the turnover of both these amines and decreases the turnover of serotonin so that the transmitter mechanism subserving the effect of l-dopa on word learning is unclear. Schuck et al. (2002) reported that in 12 healthy male volunteers, episodic memory (immediate and delayed free recall tasks) improved after the administration of piribedil (3 mg i.v.), a DA receptor agonist acting on D2 and D3 receptors. However, piribedil is also an antagonist at alpha-2-adrenoreceptors, a factor that may have contributed to its influence on cognition (Millan et al., 2001). In studies using apomorphine (Apo), a direct DA receptor agonist, Friston et al. (1992) found that Apo (5 and 10 ␮g/kg sc) impaired the free-recall performance of 24 healthy male subjects on a word list memory test. Using positron emission tomography (PET), these investigators showed that this effect was associated with an attenuation of prefrontal dorsolateral brain activity. In medicated patients with melancholic depression (N = 7) and controls (N = 5), Apo (2.5 mg/subject sc in the presence of domperidone) had no effect on cognitive task performance including the Rey Auditory Verbal Learning Task (Austin et al., 2000). In studies with DA receptor antagonists, chlorpromazine (D2 antagonism > D1) in doses of 12.5 and 25 mg had no effect on free recall and word completion tests (Danion et al., 1992) whereas haloperidol (D2 antagonism  D1) in a dose of 3 mg p.o. had a detrimental effect on immediate and delayed free recall tests (Rammsayer, Rodewald, & Groh, 2000). Neither chlorpromazine nor haloperidol are selective for DA receptors, both bind to adrenergic receptors amongst others. At this point our understanding of the role of DA in episodic memory in man is limited by several factors. Such factors include the paucity of pharmacological studies, the subject population investigated (patients or normal subjects), selectivity of agents used to address DA function, and possible adverse action of drug side effects on memory performance. Also, some of the standardized memory tests used in previous studies may not have been optimal for the detection of subtle deficits. Given the density of DA receptors in the prefrontal cortex, memory measures that are thought to rely more heavily on prefrontal cortical functions may be more effective at detecting subtle between group differences. For example, source memory tasks, where subjects must retrieve (recollect) additional contextual information in order to make correct source recognition judgments, have been shown to rely on prefrontal cortical regions more than item memory tasks (Janowsky, Shimamura, & Squire, 1989; Mayes & Daum, 1997). It is thus possible that measures of source memory would be more affected by an acute change in dopamine neurotransmission than measures of item memory. Similarly, other episodic memory tests, such as the AB–AC paired associate (memory interference) test and the categorized words test contain measures that have been shown to be sensitive to prefrontal cortical functioning (Gershberg & Shimamura, 1995;

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Shimamura, Jurica, Mangels, Gershberg, & Knight, 1995). As such, these tests may be more informative about the relationship between DA and episodic memory than are other more traditional standardized memory tests. In the present study, we evaluated the relationship between dopaminergic function and episodic memory using Apo (Lal, 1988). Apo is a highly selective agonist for D1-like (D1, D5) and D2-like (D2, D3, D4) receptors in animals (Seeman & Van Tol, 1994) and man (Tsang & Lal, 1977). Apo activates DA receptors at doses that do not affect norepinephrine (Butcher & Anden, 1969) or serotonin turnover (Lal, Sourkes, Missala, & Belendiuk, 1972). Apo stimulates both pre and postsynaptic DA receptors (Carlsson, 1977; de la Fuente-Fernandez et al., 2001). In man, Apo at low doses (3.5–5 ␮g/kg sc) (dose expressed as the salt, Apo HCl) stimulates presynaptically located DA receptors and thereby decreases DA release into the synaptic cleft, and hence, DAergic neuro-transmission whereas at doses of 7 ␮g/kg or higher Apo stimulates postsynaptically located DA receptors and increases DAergic neurotransmission (Lal et al., 1989). We hypothesized that if DA function plays a role in episodic memory, then administration of a presynaptic dose of Apo would impair performance on tests of episodic memory. We did not test to see if postsynaptic doses of Apo would enhance episodic memory because such doses, especially 10.5 ␮g/kg, induce uncomfortable side effects (Lal et al., 1989) and hence adversely affect test performance. Also, we hypothesized that given the high concentration of DA receptors in the prefrontal cortex, DA function might subserve other prefrontal cognitive functions and therefore show impairment when DA neurotransmission is decreased. We report our findings on the effect of Apo HCl (5.0 ␮g/kg sc) in healthy volunteers on three episodic memory tests (Source and item recognition test, Memory interference test, Categorized words test) and other cognitive tests thought to be sensitive to frontal lobe functions (Stroop Test, Go/no-Go, Trail Making Test, and Verbal Fluency). We expected to observe significant group by memory condition interactions on all three of our memory tests. More specifically, we expected the administration of Apo to lead to lower performance on source recognition relative to item recognition, memory interference relative to no memory interference, and on recall of categorized words relative to noncategorized words. With regards to the other cognitive tests, we expected measures of prefrontal function to be disrupted by Apo but not placebo. 2. Method A double blind, placebo-controlled, parallel group study with planned randomization and stratification by gender to ensure equal numbers and sex distribution per group was undertaken.

2.1. Subjects Twenty right-handed, healthy, paid volunteers (10 men and 10 women) participated in the study (Table 1). Selection criteria were: (1) no past or present psychiatric disorder as assessed with the Structured Clinical Interview for DSMIV-TR, Non-Patient version (SCID-I NP) (First, Spitzer, Gibbon, & Williams,

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Table 1 Subject characteristics 1 week prior to apomorphine testing Characteristic

Apomorphine (n = 10)

Placebo (n = 10)

Mean

S.D.

Mean

S.D.

Demographic characteristics Age Female (%) Education (years)

25.8 50 16.1

6.49

6.14

0.32

1.72

28.7 50 15.9

2.30

0.83

Cognitive characteristics Rey complex figure Immediate recall Delayed recall Recognition

23.35 22.85 0.73

5.36 5.81 0.18

20.9 21.5 0.72

5.54 4.76 0.13

0.33 0.48 0.91

55.1 11.0 0.57

6.0 1.81 0.13

62.6 13.2 0.65

6.04 1.31 0.17

0.01 0.01 0.23

RAVLT Correct total trials 1–5 Delayed recall Delayed recognition (accuracy)

Analysis (p)

RAVLT = Rey Auditory Verbal Learning Test. 1998), (2) no first-degree relatives diagnosed with a psychiatric/neurological disorder, (3) no significant current or past medical illnesses including history of asthma or allergy to sulphites as determined by history, physical examination and laboratory tests (electrocardiogram, hemogram, routine biochemistry, urinalysis) (4) no prior exposure to Apo, (5) on no medication, (6) no history of alcohol or drug abuse, (7) non-smoker or abstinent for 1 week, and (8) not currently pregnant. All subjects gave written consent prior to the study. The project was approved by the Ethics Committee of the Douglas Mental Health University Institute in compliance with the McGill University Guidelines for Research with human subjects.

2.2. Procedure One week prior to the testing day, subjects participated in a verbal memory test, the Rey Auditory Verbal Learning Test (RAVLT) (Lezak, 1995), and a nonverbal memory test, the Rey Complex Figure Test (Osterrieth, 1944), to assess baseline differences in cognitive ability. On the day of the experimental session, subjects arrived at the Clinical Research Unit at 9:00 h. Subjects lie down and read the information sheet following which vital signs were taken. At 9:20 h subjects received either Apo HCl 5 ␮g/kg or placebo (physiological saline) sc. Following injection, subjects remained in the semi-recumbent position for the first 10 min to minimize potential side effects. At 9:30 h subjects participated in a battery of cognitive tests which lasted 1.5 h and was administered in a fixed order for all subjects (Fig. 1). Individuals administering the injection, monitoring the procedure and administering the cognitive tests were blind as to the treatment code. Side effects which may occur after Apo (e.g. drowsiness, tiredness, nausea) were evaluated every 15 min using 10-point rating scales.

2.3. Apomorphine preparation Apo-GO ampoules containing Apo HCl 10 mg/ml in aqueous solution with sodium metabisulphate as an antioxidant were purchased from Britannia Pharmaceuticals Ltd. The solution was diluted to 1 mg/ml Apo HCl in physiological saline before use. Following sc injection, the distribution half-life is 5 min and the elimination half-life is 33 min (Gancher, Woodward, Boucher, & Nutt, 1989).

2.4. Cognitive testing The testing was conducted in a quiet room. Subjects sat comfortably in an upright position approximately 50 cm away from a laptop monitor. The center of the monitor was aligned with each subject’s eyes. Two computerized experimental tests (source/item recognition and Go/no-Go) were used.

2.5. Memory tasks 2.5.1. Source and item recognition test Stimuli consisted of 80 Snodgrass line drawing images. During an encoding phase, 64 images were presented sequentially and in a random order, with half appearing on the left side of the screen and the other half appearing on the right side of the screen. A fixation cross was continuously presented in the middle of the screen facilitating the identification of each image as being either on the left or right. Subjects were instructed to study the images and try to remember which images they saw and on which side of the screen they appeared. A recognition task followed and was divided into two subtests, a source recog-

Fig. 1. Schematic representation of the schedule of the experimental session.

A. Montoya et al. / Neuropsychologia 46 (2008) 292–300 nition test and an item recognition test, which were counterbalanced based on the participant’s subject number. In the source recognition test, 32 images (16 left and 16 right images) that had been studied during the encoding task were presented. These images were presented in the middle of the screen and subjects had to indicate with a mouse click whether they had seen the images on the left or right side of the screen. The item recognition test consisted of 32 images, 16 of which were images that had previously been presented during the encoding task and another 16 that had never been presented. The images appeared in the middle of the screen and subjects had to indicate if the images were old (studied before) or new (never studied before). For both the encoding and recognition trials, the images appeared on the screen for 4 s. The percentage of items correctly recognized in both conditions was taken as the measures of performance. 2.5.2. Memory interference test An AB-AC paired-associate test was used to study proactive memory interference. In this test, subjects were orally presented a list of 20 word pairs (e.g. A1–B1, A2–B2, etc.). The words making up each pair were matched in terms of their length and frequency. Subjects had been instructed to remember the words as pairs because their memory for the pairs would be tested immediately after. Following the presentation of the 20 word pairs, subjects were presented with the first cue word from each pair (e.g. A1) and asked to recall the second word that had been associated with it (e.g. B1). Each subject was presented the word list three times and their responses were recorded each time. Following the three trials, subjects were presented with a second list of 20 word pairs, composed of all the first words from the first list paired with 20 new words (e.g. A1–C1, A2–C2, etc.). Again, the list was orally presented and subjects, given the first cue word, had to produce the second word. The number of words recalled on trial 1 (no interference condition) and the number of new words recalled (interference condition) was analyzed. 2.5.3. Categorized words tests Three lists of 16 words were orally presented to subjects. The first list (A) was composed entirely of unrelated words. In the second one (B list), words belonging to four semantic categories (e.g. fruits, clothes, etc.) were presented randomly. In the third one (C list), words from four new semantic categories were read sequentially (e.g. horse, lion, tiger, mouse, whisky, wine, etc.). Following the presentation of each list, subjects were instructed to repeat as many words as they could recall. Organizational strategies were assessed by measuring the extent to which the subjects clustered items by category, serial order, or by subjective organization at recall. As a measure of performance, the number of words recalled correctly under the three conditions was computed.

2.6. Frontal lobe function tests 2.6.1. Go/no-Go task The Go/no-Go task is a frequently used measure of inhibitory control. Participants are asked to respond quickly to one kind of stimuli (go stimuli), but to refrain from responding to another kind of stimuli (no-go stimuli). This task was designed in our lab and modeled after tasks appearing in other studies (Bates, Kiehl, Laurens, & Liddle, 2002). Briefly, stimuli were sequentially presented in the middle of a white computer screen for 250 ms at an average rate of one every 1.75 s. Several No-go stimuli (the letter “K”) were presented randomly among Go stimuli (the letter “X”). As a measure of performance, percentage of hits (response to X and no response to K) and false alarms (response to K) were computed. 2.6.2. The stroop color-word paradigm In the Stroop test (Stroop, 1935), subjects had to use selective attention and inhibition to identify the ink color in which the words ‘red’, ‘green’, and ‘blue’ are printed. Naming the ink color of an incongruently colored word requires suppression or inhibition of the currently irrelevant word dimension. Thus, the magnitude of the Stroop interference effect is considered a measure of the inhibitory processes involved in selective attention. The time needed to complete each condition (word, color, word-color interference) was recorded and analyzed.

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2.6.3. Trail making test (TMT) This test is a measure of visuomotor coordination in which subjects are asked to connect digits (TMT-A) and digits and letters alternately (TMT-B) in proper order (Reitan, 1958). Trail A, in which subjects were instructed to connect a series of numbers by pencil line as quickly as possible with no mistakes, was used as a measure of attention. Trail B, in which the patient is instructed to connect a series of numbers and letters by pencil line as quickly as possible with no mistakes, was used as a measure of executive function. The scores were calculated as the total time in seconds required for Parts A and B of the test. 2.6.4. Verbal fluency Verbal fluency was assessed using phonemic and semantic verbal fluency, as measured by an individual’s ability to generate words beginning with a specific letter (e.g. F, A, and S) or semantic category (e.g. animals). These tests are described in greater detail elsewhere (Tombaugh, Kozak, & Rees, 1999). As a measure of performance the sum of all admissible words for the three letters and for animals was computed.

2.7. Data analysis To test the hypothesis of an association between DA activity and episodic memory, we performed a multivariate analysis with group (Apo, placebo) as an independent variable and behavioral performance on the different tests as dependent variables. When group differences were statistically significant at p < 0.01, post hoc analyses were used to determine which tests contributed to the group effect. When we discovered that there was an imbalance in our baseline measures, we adjusted for baseline characteristics using multivariable (regression) techniques (analysis of covariance). All data were analyzed using SPSS for Windows Version 11.5.

3. Results All 20 subjects completed the study. One subject reported transient light-nausea and dizziness after receiving Apo. This subject was able to complete all the required tests. The remaining subjects reported no significant adverse effects during the course of the experimental session. In addition, there were no significant differences between groups on the 10-point rating scales completed throughout the experiment (mean drowsiness: Apo = 1.99, range 0–4, control = 1.27, range 0–3; mean tiredness: Apo = 2.41, range 0–5, control = 1.10, range 0–4; mean nausea: Apo = 0.57, range 0–5, control = 0.10, range 0–2; all ps > 0.08). 3.1. Cognitive measures 3.1.1. Baseline neuropsychological performance The performance of subjects from both groups (Apo and placebo) prior to the experimental drug condition is shown in Table 1. We found baseline group differences for measures of immediate and delayed recall on the RAVLT. Subjects who later received Apo recalled fewer words during trials 1–5 and during the delayed recall than subjects who later received placebo. In order to study the effect of these imbalances on our memory measures after treatment (measures of source/item recognition and interference memory), we repeated our analyses and included these variables as covariates (analysis of covariance). After controlling for the pre-test differences there were still significant differences between groups on the source/item recognition and interference memory tests.

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Table 2 Mean ± S.D. performance of the apomorphine and placebo groups on the recognition memory and other neuropsychological tasks Apomorphine (n = 10)

Placebo (n = 10)

p Value

Memory Source recognitiona Item (new and old) Source (left and right)

85.60 ± 11.03 71.45 ± 13.92

93.70 ± 4.79 83.60 ± 12.39

0.05* 0.05*

Memory interferenceb AB-AC Trial 1 AB-AC Recall AC

12.50 ± 4.72 11.20 ± 3.70

16.70 ± 2.31 15.70 ± 3.23

0.02 0.01

6.40 ± 2.46 6.60 ± 1.90 9.40 ± 2.46

6.70 ± 2.21 8.70 ± 2.11 10.60 ± 2.72

0.79 0.03 0.31

Executive functions Go/no-Go Correct hits False alarm Go reaction time (ms)

83.90 ± 5.57 3.80 ± 3.52 415 ± 49

86.60 ± 3.56 3.20 ± 3.67 435 ± 60

0.21 0.71 0.60

Trail making Test A (s) Trail making Test B (s)

27.15 ± 5.02 57.30 ± 16.48

25.70 ± 9.13 53.70 ± 14.83

0.67 0.62

Verbal fluency FAS Animal naming

37.89 ± 9.49 19.33 ± 5.89

46.30 ± 13.94 26.40 ± 10.64

0.15 0.10

32.50 ± 7.95 21.90 ± 5.57 47.90 ± 7.73

27.30 ± 4.55 19.60 ± 2.99 44.80 ± 7.39

0.09 0.26 0.37

Categorized wordsb List A List B List C

Stroop test (s) Condition 1 Condition 2 Condition 3 a

Percentage of correct items. Total number of correct items. * p Value after adjustment: p value for item recognition after adjustment for RAVLT correct total trials 1–5, p = 0.047; p value for Source recognition after adjustment for RAVLT delayed recall, p = 0.033. b

3.2. Treatment effects on neuropsychological performance 3.2.1. Memory performance We found a treatment effect of Apo on two memory tests, the item/source recognition test and the interference memory test (Table 2). A Group × Memory condition (source versus item) ANOVA revealed a significant main effect of Group (F(1, 18) = 6.81, p = 0.02) and Memory condition (F(1, 18) = 15.43, p = 0.001), but no significant Group × Memory condition interaction (F(1,18) = 0.43, n.s.). Subjects who received Apo had lower scores on item recognition trials (85 ± 11% versus 93 ± 5%) and source recognition trials (71 ± 14% versus 84 ± 12%) than subjects who received placebo. Similarly, there was no significant Group × Memory condition (interference versus no interference) interaction for the AB–AC paired associate test (F(1, 18) = 0.053, n.s.), though there was a significant effect of Group (F(1, 18) = 8.73, p = 0.01) and a trend for an effect of Memory condition (F(1, 18) = 3.13, p = 0.09). Finally, we performed a Group × Memory condition (ANOVA on the results of the categorized words test). We found a significant main effect of Memory condition (categorized versus non-categorized words) (F(1, 18) = 36.04, p < 0.001), but no main effect of Group (F(1, 18) = 0.63, n.s.) and no Group × Memory condition interaction (F(1, 18) = 0.61, n.s.). As seen in Table 2, all subjects

performed better on the categorized word trial than the random word trial. 3.2.2. Executive functioning and other cognitive abilities performance Subjects under the influence of Apo had lower performance on all remaining tasks (Go/no-Go, Stroop, Trail Making Test, Verbal fluency) than subjects under placebo, though none of these numerical differences were statistically significant. Means and standard deviations are presented in Table 2. It can be noted from Table 2 that the reaction time in the Go/no-Go task was numerically faster in the Apo group although this effect was not significant. Other measures sensitive to response speed (e.g. Trail A, Stroop word reading) were numerically faster in the placebo group. Again, this difference was not significant. 4. Discussion The present study aimed to assess the effects of a DAergic challenge on episodic memory tests and other tests thought to be sensitive to prefrontal cortical functioning in a group of young, healthy volunteers. Evidence that memory could be affected by acute changes in DA transmission was first observed in studies of working memory. These studies have generally found

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working memory to be impaired following the administration of DA receptor antagonists and improved following the administration of DA receptor agonists (see Barch, 2004 for a review). In the current study, we hypothesized that episodic memory would be affected by a DAergic challenge in a similar way. Specifically, we expected that a decrease in DA neurotransmission would decrease performance on tests of episodic memory and that measures that relied more heavily on prefrontal cortical functions would be affected to a greater extent, given the high concentration of DA receptors in this brain region. We used the DA receptor agonist Apo at a dose (5 ␮g/kg sc), which preferentially stimulates presynaptic DA receptors and impairs DA neurotransmission (Lal et al., 1989). Hence, we expected this procedure to modulate episodic memory performance and disrupt performance on several neuropsychological measures thought to depend on prefrontal DA functioning. Interestingly, administration of Apo did have an effect on two of our memory tests, though the expected group by memory condition interactions were not observed. More specifically, administration of Apo led to decreased performance on source and item recognition tasks and on free recall trials of an AB–AC paired associate test compared to placebo. However, subjects’ performance on the source recognition task was not more affected by the active compound than was their performance on the item recognition task, nor was the AC recall disrupted to a greater extent than the first trial of the AB recall. Also contrary to our expectations, the DA challenge had little effect on a categorized words test and on other measures assessing prefrontal functions. To date, relatively few studies have examined the effect of DAergic agents on episodic memory and the results have been equivocal. In keeping with our findings, Friston et al. (1992) showed that a similar low dose of Apo (5 ␮g/kg sc) had a detrimental effect on episodic memory. However, in their study a dose of 10 ␮g/kg sc also impaired episodic memory performance. The reasons as to why the expected interactions and impairment on measures of prefrontal functions were not observed are not entirely clear. While we did not directly measure changes in DA neurotransmission in the prefrontal cortex, this dose of Apo, i.e. 5 ␮g/kg is known to inhibit DA function and that this same dose has been shown to attenuate memory-related prefrontal activity (Friston et al., 1992). Our results, regardless of significance level, were numerically in the direction of attenuation of performance. It is possible then that our memory and cognitive measures were not sensitive enough to these neuromodulatory effects of DA in the prefrontal cortex. For the most part this also seems unlikely given the wealth of evidence supporting the sensitivity of these measures to executive or prefrontal functioning. Each of our memory tests contained measures that have been shown, by lesion and neuroimaging studies alike, to be dependent on prefrontal integrity. For instance, source recognition memory tasks that are similar to the one used in the current study have been shown to be performed poorly by patients with frontal lobe lesions (Mayes & Daum, 1997) and have been found to activate left prefrontal regions relative to item recognition tasks (Rugg et al., 1999). Shimamura et al. (1995) administered the AB–AC paired associate test to a group of frontal lobe lesion patients and found that their recall of new words (“C” words) was

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disproportionately worse than the recall performance of controls. In addition, an fMRI study by Henson, Shallice, Josephs, and Dolan (2002) used a similar paired associate test in a group of healthy subjects and found clear effects of proactive interference in the prefrontal cortex. Similar results have been demonstrated for the categorized words test (Gershberg & Shimamura, 1995), as well as our other cognitive tasks (Garavan, Ross, & Stein, 1999; Ravnkilde, Videbech, Rosenberg, Gjedde, & Gade, 2002), suggesting that perhaps sensitivity to prefrontal functioning was not the main reason for the absence of an effect on performance in the Apo group. That being said, we cannot exclude the possibility that Apo elicited a response in different prefrontal regions from those recruited by our cognitive tests. It is worth mentioning that the recruitment of several other brain areas, in addition to the prefrontal cortex, is required for an optimal performance on the memory and executive function tests used in this study. Furthermore, our memory measures likely recruited some regions not recruited as strongly by the other executive tests. An example of such a region would be the hippocampus, a medial temporal lobe region known to play a critical role in episodic memory (Squire, Stark, & Clark, 2004). Hippocampal activation is commonly observed during episodic memory encoding and retrieval (Lepage, Habib, & Tulving, 1998; Schacter & Wagner, 1999). In contrast, little involvement of the hippocampus has been demonstrated in executive tests such as the Stroop test, verbal fluency test, Go/no-Go, and TMT, which involve instead mainly prefrontal, parietal and anterior cingulate cortices (Liddle, Kiehl, & Smith, 2001; Moll, de Oliveira-Souza, Moll, Bramati, & Andreiuolo, 2002; Ravnkilde et al., 2002). While little is currently known about the relationship that exists between the hippocampus, DA and episodic memory, it has recently been suggested that the hippocampus and midbrain structures form a functional loop that regulates the entry of information into long-term memory (Lisman & Grace, 2005). In this model, novelty-dependent firing within the ventral tegmental area stimulates DA release in the hippocampus, thus enhancing long-term potentiation (LTP), a form of synaptic plasticity thought to be the cellular basis of learning and memory (Malenka & Nicoll, 1999). Support for this model was recently provided by Wittmann et al. (2005) who used a reward anticipation paradigm to study the relationship between brain activity in DAergic midbrain areas and long-term memory in a group of healthy subjects. Reward-predicting pictures were associated with greater fMRI activity in midbrain regions and were recognized more accurately than neutral pictures during a delayed recognition memory test. Furthermore, the authors found greater activity in both midbrain areas and the posterior hippocampus for reward-predicting pictures that were later remembered relative to those subsequently forgotten. This finding of an association between hippocampal and DAergic midbrain activity supports the hypothesis that DAergic neuromodulation enhances longterm memory formation and may help explain why the DA challenge in the current study impaired subjects’ performance on tests of episodic memory but not their performance on the tests of prefrontal functions. Animal studies have shown that DA antagonists can block LTP in the hippocampus and consequently impair learning and memory (Morris et al., 2003)

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and our results suggest that a similar mechanism may occur in humans. An additional factor that may have had a significant influence on the observed pattern of results was the fixed order of the tests. This may have had the greatest impact on the categorized words test, which was always the first test performed, administered 10 min following Apo administration. Given Apo’s pharmacokinetic properties, it is possible that the drug exerts a significant effect only 15 min after injection. Hence, an order effect could explain the lack of significant group differences in performance on this test. It is unlikely that sedative results of Apo influenced the results. Thus, we found that reaction times and speed of responses did not significantly differ between the Apo and placebo groups. In addition, there were no significant differences between groups on subjective ratings of drowsiness or tiredness. It is possible that side effects of Apo accounted for the decreased performance in the Apo group even though the dose of Apo was quite small (emetic dose ∼85 ␮g/kg versus current dose 5 ␮g/kg). However, there was no statistically significant difference in side effects between Apo and placebo group. The peak Apo plasma concentration occurs 7.8 min after sc administration (Gancher et al., 1989), which roughly corresponds to the onset of side effects (Lal, Guyda, & Bikadoroff, 1977), which usually disappear within 45–50 min of injection. The elimination half-life of Apo is 33 min (Gancher et al., 1989) (i.e. 94% eliminated 132 min after injection, Benet, Mitchell, & Sheiner, 1990). If impaired performance were related to side effects, one would have expected impairment in performance in tests administered soon after peak Apo concentrations and little or no side effects in tests performed 70 min or more after injection. In the present study, there was no significant impairment in tests performed 10–55 min after Apo injection whereas significant impairment was noted in the source/item recognition and memory interference test which were administered 55 and 75 min, respectively, after injection. This study has several limitations. First, our sample size was very small and as such our results would need to be replicated with a larger number of subjects. Second, the protocols for the current study called for only a single dose of Apo, thus precluding the generation of dose–response curves. Third, some baseline differences in our sample were found before the randomization, specifically on measures of immediate and delayed recall on the RAVLT, although the comparability was considered unaffected. Randomized clinical trials provide the most reliable evidence of treatment effectiveness in clinical research. The main value of randomization is that treatment groups are on average comparable in terms of known and unknown subject characteristics. However, baseline differences in prognosis between groups may well occur, even in relatively large trials. Such differences between treatment groups arise by pure chance when treatment allocation is truly random. In 5% of cases, baseline characteristics will show a “significant” imbalance between randomized groups (p < 0.05). P values of statistical tests of imbalance cannot be interpreted as indicating whether randomization works and may only serve a descriptive purpose. An estimate of the effectiveness of treatment can statistically be

adjusted for prognostic baseline characteristics with multivariable (regression) techniques (e.g. analysis of covariance) and in our study, after controlling for the pre-test differences, there was still a significant difference between the post-test scores on source/item recognition and interference memory. Fourth, our battery of tests did not include any measures of working memory, making comparisons with other studies difficult. Finally, it would also have been interesting to gather genetic information from our subject sample given recent findings of differences in episodic memory functioning related to different polymophisms of the catechol O-methyltransferase (COMT) gene (de Frias et al., 2004), a gene coding for the enzyme that breaks down DA. Future studies on the effects of dopamine on memory would benefit from methodological improvements over the present experimental design. Firstly, the use of a repeated measurements design where each participant is his/her own control would remove several potential confounds such as group differences in cognitive abilities. Secondly, task difficulty should be set in a way to avoid potential ceiling or floor effects affecting selectively a condition of interest. Findings in this small sample of subjects suggest that DA mechanisms affect episodic memory function. These findings are consistent with studies of healthy aging and psychiatric illness, which relate impaired memory function to dysfunctions of the dopamine system (Backman & Farde, 2001; Dujardin & Laurent, 2003; Goldman-Rakic, Castner, Svensson, Siever, & Williams, 2004). A better understanding of the role of DA in episodic memory could provide important insights into how these memory deficits develop and how they may be reduced. Future pharmacological studies incorporating functional neuroimaging techniques and genetics would be well-suited to further elucidate the relationship between DA, frontal and hippocampus functioning, and individual differences in episodic memory ability. Acknowledgements This study was supported by the Canadian Institutes of Health Research (grant #53280), a Young Investigator Award from the NARSAD, and a salary award from the Fonds de la Recherche en Sant´e du Qu´ebec to ML. References Abi-Dargham, A., Martinez, D., Mawlawi, O., Simpson, N., Hwang, D. R., Slifstein, M., et al. (2000). Measurement of striatal and extrastriatal dopamine D1 receptor binding potential with [11C]NNC 112 in humans: Validation and reproducibility. Journal of Cerebral Blood Flow and Metabolism, 20, 225–243. Austin, M. P., Mitchell, P., Hadzi-Pavlovic, D., Hickie, I., Parker, G., Chan, J., et al. (2000). Effect of apomorphine on motor and cognitive function in melancholic patients: A preliminary report. Psychiatry Research, 97, 207–215. Backman, L., & Farde, L. (2001). Dopamine and cognitive functioning: Brain imaging findings in Huntington’s disease and normal aging. Scandinavian Journal of Psychology, 42, 287–296. Backman, L., Ginovart, N., Dixon, R. A., Wahlin, T. B., Wahlin, A., Halldin, C., et al. (2000). Age-related cognitive deficits mediated by changes in the striatal dopamine system. American Journal of Psychiatry, 157, 635–637.

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