Brain Indices of Nonconscious Associative Learning

CONSCIOUSNESS AND COGNITION ARTICLE NO.

6, 519–544 (1997)

CC970322

Brain Indices of Nonconscious Associative Learning P. S. Wong,*,1 E. Bernat,†, S. Bunce,† and H. Shevrin† *Department of Psychology, New School for Social Research, New York, New York 10003; and †Department of Psychiatry, University of Michigan Medical Center, Ann Arbor, Michigan Using a classical conditioning technique, this study investigated whether nonconscious associative learning could be indexed by event-related brain activity (ERP). There were three phases. In a preconditioning baseline phase, pleasant and unpleasant facial schematics were presented in awareness (suprathreshold). A conditioning phase followed, in which stimuli were presented outside awareness (subthreshold, via energy masking), with an unpleasant face (CS1) linked to an aversive shock and a pleasant face (CS2) not linked to a shock. The third, postconditioning phase, involved stimulus presentations in awareness (suprathreshold). Evidence for acquisition of a conditional response was sought by comparing suprathreshold pre- and postconditioning phases, as well as in the subthreshold conditioning phase itself. For the pre-postconditioning phase analyses, significant ERP component differences differentiating CS1 and CS2 were observed for N1, P2, and especially P3. For the conditioning phase, significant differences were observed in the 100–400 ms. post-stimulus region reflecting a CS1 processing negativity. Brain activity does indeed index the acquisition of a conditional response to subthreshold stimuli. Associative learning can occur outside awareness.  1997 Academic Press

INTRODUCTION

In the past 15 years, there has been increasing interest in the experimental investigation of conscious and nonconscious processes (Shevrin & Dickman, 1980; Kihlstrom, 1987).2 In one approach to this issue, investigators have relied on classical conditioning paradigms to explore the extent to which a response established to stimuli in awareness could be elicited at a later time when the stimuli were presented outside awareness (Lazarus & McCleary, 1951; Corteen & Wood, 1972; Dawson & Schell, 1982). These studies relied on dichotic listening paradigms (often using aversive shock as unconditioned stimulus) with electrodermal measures serving as the main index of responsivity. Other studies, particularly by Ohman and colleagues (Ohman, Dimberg, & Esteves, 1988; Ohman & Soares, 1993; 1994), have relied on visual backward masking techniques to demonstrate the same effect. Recently, we conducted a study that paralleled the Ohman et al. work by using energy masked facial schematics as conditional stimuli (CS) in a classical condition1

Address correspondence and reprint requests to: Philip S. Wong, Department of Psychology, New School for Social Research, 65 Fifth Avenue, New York, NY 10003. E-mail: [email protected]. This research was supported in part by a grant from the Ford Motor Company to H.S. We thank Michael Snodgrass, Jennifer Stuart, and William J. Williams for their assistance in various aspects of the study. Portions of this study were presented at the 1995 meetings of the American Psychological Society and American Psychological Association in New York. 2 We use the term, nonconscious, to refer to activity outside awareness that has a mental referent. Much physiological activity, of course, may not be represented mentally in any form. 519 1053-8100/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.

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ing paradigm (Wong, Shevrin, & Williams, 1994). Several methodological advances in this study were noteworthy, including careful measurement of individual visual thresholds operationalizing awareness, and the use of event-related brain potentials (ERP) as an index of the learning process. We systematically replicated previously reported results (Ohman et al., 1988) demonstrating that facial schematics conditioned in awareness could elicit an electrodermal response (SCR) when later presented outside awareness. The results from both our study and those of Ohman et al. were consistent with earlier findings based on dichotic listening, and support the conclusion that a conditional autonomic response can be elicited even when the CSs are inaccessible to awareness. Extending the SCR results, we also discovered a brain response to subthreshold presentations of the conditioned facial stimuli. Distinct slow wave activity in response to the CS1 (unpleasant face), and not to the CS2 (pleasant face), emerged in the region prior to the point at which the shock had been delivered during acquisition. This ERP activity was similar to what others have described as an expectancy wave (Simons, Ohman, & Lang, 1979), indicating that an anticipatory process could be elicited entirely outside awareness. In other words, nonconscious activity involves processes associated with expectation or anticipation—processes considerably more complex than often attributed to activity outside awareness (Greenwald, 1992). Additionally, our findings highlighted the usefulness of measuring brain activity in response to stimuli presented in and out of awareness, and added to a growing literature using ERPs and subthreshold stimuli (e.g., Brandeis & Lehmann, 1986; Libet, Alberts, Wright, & Feinstein, 1967; Kostandov & Arzumanov, 1986, 1977; Shevrin, 1973, 1988; Shevrin & Rennick, 1967; Shevrin & Fritzler, 1968; Shevrin, Williams, Marshall, Hertel, Bond & Brakel, 1992). One can conclude, from the studies using autonomic measures and from our results extending the effect into central nervous system activity, that a previously learned response can be elicited or activated by stimuli outside awareness. This conclusion has important implications not only for theories of learning, but also for theories of psychopathology. For example, these findings can serve as the basis for neural models of anxiety disorders, and how learning during aversive circumstances (i.e., with an unpleasant shock) can have lasting effects that may be elicited, outside awareness, in a seemingly automatic way. A closely related issue, and one that may have considerably more theoretical significance, is whether learning itself can occur outside awareness. Could psychopathological conditions develop from aversive circumstances during which an individual was unaware of the object (CSs) or of the link betweeen object and event? In other words, can nonconscious associations occur, and do these associations have identifiable effects over time? Studies on nonconscious learning have increased in recent years in parallel with an increased interest in conscious and nonconscious processes. Using primarily cognitive experimental techniques, investigators have revealed that learning without awareness can occur in two basic ways. First, acquisition of knowledge can occur with an individual unaware of what is being learned (e.g., Reber (1989) on artificial grammar; Lewicki (1992) on covariation in social judgment tasks; for a recent alternative view, see Shanks & St. John, 1994). Typically, these paradigms involve demon-

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strations of a person acquiring a complex set of rules without awareness of doing so, with stimuli that are themselves accessible to awareness (i.e., suprathreshold). The rule learning is implicit. Second, learning associated with subthreshold stimuli— stimuli that are inaccessible to awareness—has been demonstrated primarily with the mere exposure effect (Kunst-Wilson & Zajonc, 1980; see also Murphy & Zajonc, 1993).3 Stimulus presentations outside awareness can bias a person’s preference for the stimuli, even when the person cannot recognize the stimuli as having been presented previously. Learning in this sense is entirely outside awareness. With some exceptions, little work has been done to elucidate the neural systems that underlie either kind of nonconscious learning—the implicit, knowledge-based kind or the unconscious perceptually based kind. For example, a few investigators have discovered neural indices of incidental learning using electrophysiological approaches (Paller et al., 1987; Begleiter et al., 1967, 1969). Investigations of associative learning (using classical conditioning techniques and autonomic measures) have been far more prevalent, although the results seem not entirely consistent with the aforementioned cognitive studies. A review of this literature leads one to the conclusion that in order to acquire a differential autonomic conditional response, the CS, among other things, has to be attended to and easily discriminable (Dawson & Schell, 1985). That is, the CS needs to be in or accessible to awareness. Acquisition of a conditional response to subthreshold stimuli has yet to be demonstrated definitively (although see Esteves, Parra, Dimberg, & Ohman, 1994, for recent electrodermal evidence). The conclusion that the CS needs to be in awareness is at odds with results from purely cognitive experiments such as the subthreshold mere exposure paradigm. The present study explored the question of whether nonconscious learning (the perceptually based kind) could occur using subthreshold CSs in a conditioning paradigm. We were specifically interested in whether this kind of learning could be detected in central nervous system activity. Such information would facilitate a more comprehensive understanding of the nature of nonconscious learning from both cognitive and neural perspectives and have implications for models of psychopathology. In structure, the present study parallels our previous study (Wong et al., 1994) and draws upon research reported by Ohman and colleagues (Dimberg, 1986; Esteves, Dimberg, & Ohman, 1994; Ohman & Dimberg, 1978; Ohman et al., 1994). One consistent finding reported by Ohman and colleagues has been the salience of angry faces in conditioning. After pairing with an aversive shock (US), angry faces have exhibited slower extinction (Dimberg, 1986) and survived backward masking (Esteves et al., 1994) when compared to happy faces paired with the US. Based on these findings, we relied on a partial-factorial design in our previous study by pairing unpleasant faces with aversive shock, to explore subsequent subthreshold conditional responses (Wong et al., 1994). The rationale was straightforward: given the subtle effects often elicited by subthreshold presentations (e.g., Shevrin et al., 1992), it seemed important to provide a strong test of whether any subthreshold response was detectable. If unpleasant faces showed no effect, then it could be concluded that even 3

The effects of subthreshold stimulus presentations, including semantic activation, has been the subject of much investigation (e.g., Holender, 1986; Greenwald, 1992). These studies often posit brief activation due to a subthreshold presentation, and are only indirectly related to learning.

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less salient stimuli would be unlikely to work. However, if unpleasant faces gave rise to an effect, then subsequent studies could explore the parameters of that effect. The present study is based on the same logic, and addresses another conceptual question—does nonconscious associative learning occur? There were three phases in the experiment. In the first preconditioning phase, facial schematics depicting pleasant and unpleasant expressions were presented in awareness (suprathreshold). These presentations established baseline activity to the CSs. In the second conditioning-acquisition phase, stimuli were presented outside awareness (subthreshold). Stimuli were linked in a differential conditioning paradigm to an aversive shock in a partial-factorial design (interstimulus interval 5 800 ms; CS1/unpleasant face; CS2/pleasant face; probability 5 0.8). In the third postconditioning phase, the stimuli were presented in awareness (suprathreshold). Evidence for the acquisition of a conditional response was sought in brain activity (ERP) when comparing the suprathreshold pre- and postconditioning phases (an indirect index of acquisition), as well as in the subthreshold conditioning phase itself (a direct index of acquisition).4,5 We addressed questions in three related areas. In the Wong et al. (1994) study, P3 amplitude differences (unpleasant face . pleasant face) were observed in the suprathreshold acquisition phase. These amplitude differences reflected the acquisition process using suprathreshold stimuli, and were consistent with existing theories that P3 is an index of the relative salience of stimuli (e.g., Begleiter et al., 1983; Donchin & Coles, 1988). In the present study, although P3 would still index stimulus salience it would not reflect direct acquisition as much as subsequent processes associated with extinction. Our first hypothesis (a priori ) was that an ERP P3 component amplitude advantage would exist for CS1 compared to CS2 as a function of previous subthreshold conditioning. We expected that P3 component amplitude would remain stable for CS1 and would decrease for CS2, from pre- to postconditioning. Such evidence would be consistent with a ‘‘resistance to extinction’’ effect associated with CS1; CS2 responses, in contrast, would reflect a decrease in amplitude to a greater extent than found for CS1. A finding of differential component effects (such as P3) for CS1 and CS2 would be consistent with extinction and sensitization results reported in the literature by Ohman and others using SCR with unpleasant faces (e.g., Esteves et al., 1994). Second, would we observe any other component differences (e.g., in N1) between pre- and postconditioning phases? Researchers using suprathreshold stimuli have identified early negative component differences (N1, Nd) possibly related to attentional processes (e.g., Naatanen, 1990) primarily in the auditory sphere. No early component differences were discovered in the Wong et al. (1994) study for the suprathreshold acquisition phase, although evidence of early attentional selectivity would be consistent with nonconscious associative learning. Third, would there be systematic differences in processing CS1 and CS2 in the 4

We collected electrodermal measures; unfortunately technical problems in the data acquisition procedure invalidated the data. 5 This report focuses on early ERP activity (800 ms post-stimulus); results from facial EMG responses and other ERP activity will be reported elsewhere.

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subthreshold conditioning phase itself? Some investigators have identified N1-P2 differences associated with subthreshold stimuli (Shevrin, 1973), although no such differences were observed to previously conditioned subthreshold stimuli (Wong et al., 1994). There have been no reports in the literature, however, examining directly the effects of acquisition of a conditional response using subthreshold stimuli on ERPs. In each of the three areas identified above, positive results would provide converging evidence to support the conclusion that acquisition of a conditional response can occur when stimuli are perceptually inaccessible to awareness. METHOD

Subjects Subjects were recruited through advertisements in a university community for a psychology study on perception. Remuneration was $25 for approximately 3.5 to 4.0 h. Subjects were screened for general health and for handedness. Those with a history of neurological disorder or who were currently experiencing significant problems with their physical or emotional health were excluded. Subjects were scheduled for a laboratory appointment and were asked to come to the appointment well-rested and to refrain from drinking alcoholic beverages the evening prior to the experiment. In total, 10 subjects participated in the study. All subjects were right-handed men, with vision correctible to 20/20. The mean age was 21.6 years (SD 5 1.4). One subject was eliminated due to a high level of physiological artifact (excess muscle tension and movement) in the data. Analyses are reported for the remaining sample of 9 subjects. Procedure The overall procedure paralleled that reported by Wong et al. (1994). After a general orientation (including screening and other questionnaires), electrodes were attached for physiological recording. Aversive shock and visual threshold routines (described below) were conducted prior to beginning the main experiment. The experimental phases and data collection sequence in the main experiment are presented in Fig. 1. In the data collection sequence, a tone (T1) signaled the beginning of a trial, and the subject responded by saying ‘‘ready’’ (T2) when he was looking at the fixation point. Four to 6 s after T2 the data collection cycle began; the cycle lasted for 4400 ms. The prestimulus interval was 400 ms. S1 denotes presentation of a masked facial schematic (conditioning phase) or unmasked facial schematic (pre- and postconditioning phase). S2 denotes the shock/no-shock event, which in the conditioning phase occurred 800 ms after S1. Two tones signaled the end of the data collection cycle, with the intertrial interval approximately 10–15 s. Individual presentations were automated and required no interaction with the experimenter. During all trials, subjects were instructed to remain as still as possible, look at the fixation point, and keep eye blinks to a minimum. Subjects were told that at some point soon after saying ‘‘ready’’ there would be a quick flash of something on the screen, which might or might not be followed by a shock several seconds later. Sub-

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FIG. 1. Organization of experiment. (a) Individual trial presentation sequence. (b) Experimental phases.

jects were reminded periodically to keep looking at the fixation point and to minimize eye blinks during trials. There were three experimental phases: preconditioning, conditioning, and postconditioning. The preconditioning and postconditioning phases consisted of 48 random presentations (24 each of CS1 and CS2); the conditioning phase consisted of 72 random presentations (36 each of CS1 and CS2). In the preconditioning phase, the CSs were shown in awareness (suprathreshold) to establish baseline activity. In the conditioning phase, CSs were shown outside awareness (subthreshold) and linked in a differential conditioning paradigm to an aversive shock (ISI 5 800 ms; CS1, unpleasant face; CS2, pleasant face; probability 5 0.8). In the postconditioning phase, the schematics were again shown in awareness. At the end of the main experiment, subjects were re-tested on visual threshold, unhooked, debriefed, paid, and dismissed. Visual Stimuli, Apparatus, and Masking Technique The pleasant and unpleasant facial schematics were identical to those used in the Wong et al. (1994) study, and consisted of line drawings of faces in schematic form. Stimuli were equated for perceptual characteristics, e.g., number and width of lines, and rated on the extremes of an evaluative scale. Two equivalent sets of stimuli were used; each set consisted of one pleasant and one unpleasant schematic. One set was used in the visual threshold procedure, and the other in the main conditioning experiment, with sets counterbalanced between subjects. The stimuli were presented on 3 3 5 white cards in two fields of a three-field Gerbrands Model T3-8 tachistoscope. The CS1 and CS2 fields were counterbalanced between subjects; the third field was used as a fixation field. Field brightness was tested for luminance level and pulse width, and equated for each field. Luminance levels for the fields were 5 foot-lamberts; ambient room light conditions were approximately the same. The stimuli were circles subtending 1.9 degrees visual angle in diameter. Stimulus duration for the energy masked (subthreshold) presentations was 2 ms. Stimulus duration for the unmasked (suprathreshold) presentations was 50 ms.

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Visual Threshold Technique In our previous study (Wong et al., 1994), visual threshold was established and tested for each subject, in part to address previous criticism in the literature regarding the potential for significant between-subject variability in psychometric response functions. In the Wong et al. study we found that individual thresholds, as identified by an adaptive staircase technique, ranged from 2 to 4 ms with a mean of 2.35 ms. In the present study, we decided to forego establishing individual thresholds and instead presented masked stimuli at a duration of 2 ms for all subjects. Although this approach does not control for individual variability in response functions, we chose it for a number of reasons. First, the variability in thresholds in the Wong et al. (1994) study was relatively narrow (i.e., 2 to 4 ms). Second, since we were using identical visual presentation conditions to the earlier study, a 2-ms threshold duration would be at the low end of the expected range (which, if anything, would decrease the probability of finding positive results since some subjects presumably would have thresholds above 2 ms). And third, presenting the same threshold duration to all subjects is considerably more efficient from a procedural standpoint. Subjects were tested at the 2 ms duration in a forced-choice two-alternative task. Subjects were first shown one suprathreshold presentation of each facial schematic and then told that for each subsequent presentation he should decide whether the facial expression was pleasant or unpleasant. Subjects were told each facial expression would be presented an equal number of times in random order, that responses should be distributed equally, and to guess if uncertain about a response. The test consisted of 40 trials and was administered once before and once after the main experiment to monitor any potential changes in threshold level. The mean correct for the 40-trial pre-test was 19.77 (SD 5 4.29), and for the posttest was 19.67 (SD 5 2.74). A discordancy test for single outliers (Barnett & Lewis, 1984; Snodgrass et al., 1993) was applied to the extreme low and high values in the sample to determine whether or not a subject was performing within an expectable chance distribution. Each outlier value was assessed for discordancy relative to its immediate sample (i.e., a value in the pre-test condition was evaluated relative to the pre-test sample); in addition, each value was assessed relative to the combined pre- and post-test samples. None of the extreme values qualified for outlier status (p . .05), indicating that all subjects performed at expectable chance levels (neither too high nor too low) on the pre- and post-test trials and in the combined trials. The pre- and post-test trials also were subject to an analysis of variance, which was not significant. Subjects were carefully questioned about their subjective visual experiences during pre- and post-tests, as well as during the conditioning phase. None of the subjects reported seeing an internal feature of the circle with any certainty. ERP Measures Standard Grass Instrument silver-silver chloride electrodes were used. Prior to electrode application, sites were cleaned with a mild abrasive solution, and then affixed with Grass electrode paste. Recording sites were F3, F4, Cz, and Pz using the International (10–20) Electrode Placement System, with linked earlobes as reference and left mastoid as ground. Electrode impedance was under 10 Kohms. Eye activity

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was monitored by electrodes placed on the outer canthus and suborbital ridge of the right eye. Recordings were monitored on-line by a Grass Model 8-24, digitized at 250 Hz, and stored in computer files for off-line analysis. The high frequency cutoff was 70Hz. The Grass AC amplifiers were set to a low frequency cut-off of 0.01Hz. Individual trials contaminated by artifacts (eye blinks, muscle tension, or any suspicious activity which would render a trial unusable) were rejected by visual inspection and replaced on-line. ERP measures for the pre- and postconditioning phases were based on 24 presentations (per stimulus category for each experimental phase); the conditioning phase measures were based on 36 presentations (per stimulus category). The analyses in this report include the 400-ms prestimulus interval and the 800-ms post-stimulus interval (at which point the shock event occurred in the conditioning phase). The overall analysis, unless otherwise specified, was divided into two parts: (1) Precon (preconditioning) vs Postcon (postconditioning) and (2) Con (conditioning). The Precon vs Postcon analysis of variance involved a Phase(2; pre-post) 3 Face(2; pleasant-unpleasant) 3 Electrode(4) analysis, with the main (including a priori) hypotheses centered around a Phase 3 Face interaction. The Con analysis involved a Face(2) 3 Electrode(4) analysis of variance. Standard component measures (peak amplitude and latency, and area) were obtained on each individual subject’s averaged ERP profile for N1 (90–150 ms), P2 (160–220 ms), and P3 (248–548 ms) in all analyses. Early (100–400) and Late (400– 700) area measures were obtained in the Con analyses. An initial analysis of the pre-stimulus interval was undertaken in order to determine whether there was systematic variation in this interval which might bias subsequent analyses. Indeed, a Phase(3; pre-con-post) 3 Face(2) 3 Electrode(4) analysis of variance yielded a three-way interaction (F(6, 48) 5 2.53; p 5 .05; e 5 .7695) and a trend for the main effect of phase (F(2, 16) 5 3.48; p 5 .06). No other results approached significance. These results indicated that some variability existed in the pre-stimulus intervals, which was not surprising especially across phase. Consequently, for each subject, the ERP average for face and electrode within phase was adjusted so that the pre-stimulus interval averaged to zero. That is, the average prestimulus interval for each profile was subtracted from all post-stimulus values for that profile, effectively eliminating any pre-stimulus variability and allowing for direct comparison of component and area measures across phase, face and electrode. Aversive Shock Procedure Stimulating electrodes were attached to the distal phalanges of the index and ring fingers of the preferred hand (right). The stimuli were single 200-ms constant-current square wave pulses, delivered by a Grass Model S-88 Stimulator and completely isolated from ground by a Stimulus Isolation Unit (SIU-7). The intensity level of the stimulus was determined by each subject, and identified as the level at which the sensation felt ‘‘annoying or unpleasant.’’ Subjects were told that the sensation should not be painful in any way; in no case did the levels go beyond 5 mA. Subjects rated the degree to which the stimulus was ‘‘unpleasant’’ or ‘‘annoying’’ on a 9-point scale (9 5 high; 1 5 low) during threshold determination,

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FIG. 2. ERP grand average at each electrode: Analysis by phase (a, preconditioning; b, postconditioning). y axis (at stimulus onset) 5 4 µv increments; x axis 5 400-ms intervals; solid line, CS1; dotted line, CS2.

as well as after the conditioning experiment. These threshold methods parallel those used by Wong et al. (1994). The mean pre-test rating was 5.78 and the mean post-test rating was 3.8. An analysis of variance was significant (F(1, 8) 5 10.29; p 5 .01), indicating that a decrease in shock unpleasantness occurred over time. This result is likely associated with an habituation effect. RESULTS Preconditioning vs Postconditioning Phase

The grand average ERP profiles are presented in two ways for purposes of comparison. Figures 2a and 2b are an analysis by phase; activity in response to the CS2/ pleasant is contrasted with CS1/unpleasant in the (a) Precon phase and (b) Postcon phase. On inspection of this figure, the most obvious differences emerge at electrodes Cz and Pz in the Postcon phase (Fig. 2b). Here, activity in response to CS2 is lower in amplitude than CS1 from stimulus onset until just prior to where the shock/noshock event occurred in the subthreshold conditioning phase. No such differences

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FIG. 3. ERP grand average at each electrode: Analysis by stimulus (a, CS1/unpleasant; b, CS2/ pleasant). y axis (at stimulus onset) 5 4 µv increments; x axis 5 400-ms intervals; solid line, preconditioning; dotted line, postconditioning.

emerge in the Precon phase. Figures 3a and 3b are an analysis by stimulus; activity in response to the CSs across Precon and Postcon phases is examined for (a) CS1/ unpleasant and (b) CS2/pleasant. On inspection of this figure, clear differences emerge at electrode Cz and Pz for the CS2/pleasant stimulus (Fig. 3b). Here, activity in response to CS2/pleasant is lower in amplitude in the Postcon phase compared to the Precon phase. Activity in response to the CS1/unpleasant does not change across phase. Further elaboration of the methods applied to assess the statistical significance of the ERP grand average differences in the context of our experimental hypotheses are described below.6 Table 1 includes values for all component measures. Main Component Findings P3 Amplitude. Peak amplitude of the P3 component was subjected to a Phase(2) 3 Face(2) 3 Electrode(4) analysis of variance. There was a significant main effect of Electrode (F(3, 24) 5 5.94; p 5 .029; e 5 .4213; Pz . Cz . F3-F4) and a All statistical tests reported regard a significance level of p , .05 (two-tailed) as consistent with rejection of the null hypothesis. For repeated measures analyses, the Huynh-Feldt correction procedure is applied with epsilon reported. 6

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TABLE 1 Component Mean (SD) Values Amplitude (µv)

Latency (mseconds)

Area (µv/region)

CS1

CS2

CS1

CS2

CS1

CS2

22.0 (3.2) 20.9 (2.8)

2.9 (3.0) 24.2 (5.3)

120 (26) 125 (22)

116 (25) 122 (16)

28 (38) 30 (43)

44 (45) 27 (77)

6.4 (3.2) 6.3 (4.4)

6.6 (4.5) 4.5 (2.6)

185 (18) 194 (20)

183 (24) 193 (24)

48 (44) 48 (66)

49 (79) 14 (45)

12.8 (7.2) 13.1 (9.1)

14.7 (8.6) 11.9 (6.3)

364 (28) 414 (60)

372 (46) 367 (36)

553 (450) 505 (480)

627 (565) 389 (311)

22.0 (1.4) 23.0 (2.0)

21.9 (3.0) 24.2 (4.0)

117 (26) 130 (16)

118 (19) 118 (18)

21 (30) 1 (47)

23 (50) 210 (62)

6.4 (3.4) 6.4 (4.4)

5.8 (4.0) 5.0 (2.9)

196 (21) 196 (20)

184 (24) 184 (26)

42 (41) 41 (64)

37 (62) 14 (51)

12.1 (7.9) 11.6 (7.4)

12.7 (8.5) 11.0 (6.3)

360 (39) 390 (60)

343 (44) 357 (62)

483 (433) 394 (379)

531 (505) 311 (318)

22.2 (2.0) 23.4 (2.5)

22.7 (1.7) 22.4 (2.7)

126 (23) 115 (18)

116 (21) 120 (14)

16 (36) 24 (53)

12 (36) 7 (46)

5.8 (3.3) 4.6 (3.8)

5.0 (3.0) 4.4 (2.9)

196 (20) 198 (22)

195 (21) 180 (23)

37 (48) 23 (55)

25 (36) 20 (43)

7.4 (5.8) 7.4 (4.2)

8.1 (4.9) 7.7 (4.2)

351 (57) 392 (84)

370 (80) 382 (74)

224 (360) 207 (285)

267 (328) 192 (343)

22.6 (2.4) 24.2 (1.9)

22.8 (1.7) 23.2 (4.0)

124 (26) 117 (18)

120 (23) 122 (12)

16 (35) 27.9 (38)

7 (32) 21 (53)

6.3 (3.0) 4.6 (3.3)

6.0 (3.2) 4.9 (3.4)

187 (18) 196 (21)

192 (21) 194 (25)

41 (44) 27 (48)

37 (39) 25 (58)

7.9 (4.8) 7.7 (3.5)

7.9 (4.1) 7.9 (4.1)

359 (63) 338 (59)

352 (89) 341 (64)

199 (330) 172 (271)

238 (279) 178 (333)

Pz N1 Precon Postcon P2 Precon Postcon P3 Precon Postcon Cz N1 Precon Postcon P2 Precon Postcon P3 Precon Postcon F4 N1 Precon Postcon P2 Precon Postcon P2 Precon Postcon F3 N1 Precon Postcon P2 Precon Postcon P3 Precon Postcon

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significant Phase 3 Face 3 Electrode three-way interaction (F(3, 24) 5 4.56; p , .011; e 5 1.000). Post-hoc test 7 of relevant contrasts revealed that the most notable contribution was at Pz, where CS2 amplitude was significantly smaller in the Postcon phase than in the Precon phase (F(1, 8) 5 7.82; p 5 .02), whereas CS1 amplitude did not change (F(1, 8) 5 .16; n.s.). No other effects were observed within electrode although the means are all in directions consistent with Pz. N1 Amplitude. Peak amplitude of the N1 component was subjected to a Phase(2) 3 Face(2) 3 Electrode(4) analysis of variance. A highly significant Phase 3 Face 3 Electrode three-way interaction emerged (F(3, 24) 5 13.34; p , .001; e 5 .6396). Post-hoc tests of the relevant contrasts revealed that the most notable contribution to the interaction occurred at electrode Pz, where CS2 amplitude was more negative than CS1 in the Postcon (F(1, 8) 5 4.34; p 5 .07) but not in the Precon (F(1, 8) 5 .66; n.s.). At Cz, there was an overall phase effect, where activity in the Postcon was more negative than Precon for both CS1 and CS2 (F(1, 8) 5 6.62; p 5 .03). No other effects within electrode were observed although the means were in directions consistent with Pz. P2 Amplitude. Peak amplitude of the P2 component was subjected to a Phase(2) 3 Face(2) 3 Electrode(4) analysis of variance. A marginally significant Phase 3 Face 3 Electrode three-way interaction emerged (F(3, 24) 5 2.72; p , .076; e 5.8802). Inspection of the relevant cell means revealed that the most notable effects were at Pz, with the amplitude of the CS2 increasing from Precon to Postcon, while the amplitude of the CS1 remained the same. Summary of main component findings. The P3 amplitude findings indicated that activity associated with the CS2 was smaller (less positive) in the Postcon phase compared to the Precon phase, whereas CS1 did not change (and in fact became slightly larger in the Postcon). The effects are strongest at Pz and Cz. These P3 findings are highly consistent with our first hypothesis: that we would find a P3 component amplitude advantage of CS1 over CS2. The findings support the conclusion that P3 changes systematically as a function of previous subthreshold conditioning. The N1 amplitude findings indicated that activity associated with the CS2 was more negative than activity associated with the CS1 in the Postcon phase but not in the Precon phase. These effects were most prominent at Pz. The P2 amplitude findings indicated that P2 activity was smaller (less positive) for the CS2 than for the CS1 in the Postcon phase, with no difference in the Precon phase (although specific P2 contrasts could not be tested statistically because the relevant interactions reached only marginal significance levels). Taken together, the N1 and P2 amplitude findings shed light on our second area of inquiry: previous subthreshold conditioning appears to affect not only the P3 component, but also N1 and P2 component activity. Corollary Component Findings Analyses of component latencies for N1, P2, and P3 also were performed. Although some statistical trends were observed, there were no consistent latency differences either within or across components. 7

All post-hoc contrasts were adjusted using the Scheffe-type method (O’Brien & Kaiser, 1985).

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Analyses of component areas for N1, P2, and P3 were performed in parallel with the component amplitude analyses. Area results were consistent with the component amplitude findings, highlighting the differences in component responses across preand postconditioning phases. Summary of Preconditioning vs Postconditioning Phase Findings The results from the Preconditioning vs Postconditioning phase analyses bear on several questions set forth in the introduction to this paper. The first question concerned whether we would observe component differences associated with the conditioning process; it was predicted that P3 amplitude would reflect the conditioning process across pre- and postconditioning phases. This prediction was confirmed: P3 amplitude associated with the CS2 came significantly smaller (less positive) in the Postcon phase compared to the Precon phase, whereas CS1 did not change from Precon to Postcon, and was larger than CS2 in the Postcon. This finding supports the general experimental hypothesis that acquisition of a conditional response can occur with subthreshold stimuli. Upon closer inspection of the grand average profiles (Figs. 4 and 5 for Pz), we observed in greater detail how the experimental effect was expressed. In Fig. 4 (the analysis by phase), the CS2 profile is more negative than the CS1 in the Postcon phase (Fig. 4b) but not in the Precon phase (4a). This reflects an overall decrease in the ERP amplitude for CS2, from stimulus onset to approximately 800-ms poststimulus. Similarly, in Fig. 5 (the analysis by stimulus), the CS2 profile decreases notably from Precon to Postcon phases (Fig. 5b), whereas the CS1 does not (Fig. 5a). Taking these observations into account, the effects observed for the N1, P2, and P3 components combined are highly consistent with each other. That is, for CS1, we observe a smaller (less negative) N1, a larger (more positive) P2, and a larger P3, with CS2 having opposite results. These results suggest that the component effects observed may be contributing to a generalized decrease in amplitude for CS2. This result is consistent with our main hypothesis. One effect of the acquired conditional response is that the CS1 maintains its activation (as reflected, for example, in P3 component differences) while CS2 decreases substantially. This result is consistent with the ‘‘resistance to extinction’’ effect to particularly salient stimuli (e.g., Ohman & Soares, 1993). In contrast to the CS1 activity, CS2 activity decreases through repetition in the Postcon phase compared to the Precon phase. Conditioning Phase

The grand average ERP profiles for the Conditioning phase of the experiment are presented in Fig. 6. On inspection, the most salient differences emerge in electrodes Cz and Pz. Soon after stimulus presentation, the CS1/unpleasant face activity becomes more negative than the CS2/pleasant face activity. The differences between CS1 and CS2 disappear shortly before the shock-noshock event occurs at 800-ms post-stimulus (at the arrow). Further elaboration of the methods applied to assess the significance of the differences observed in the ERP grand averages are described below. Component and area values are presented in Table 2.

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FIG. 4. Detailed analysis by phase for Pz (a, preconditioning; b, postconditioning). y axis (at stimulus onset) 5 4 µv increments; x axis 5 400-ms intervals; solid line, CS1; dotted line, CS2.

Component Findings Component amplitude, latency, and area measures were subjected to a Face(2) 3 Electrode(4) analysis of variance for N1, P2, and P3.8 The N1 peak amplitude analysis revealed a significant main effect of Face (F(1, 8) 5 5.91; p , .04; CS1 more negative than CS2) and a marginally significant interaction of Electrode 3 Face (F(3, 24) 5 2.96; p , .06; e 5 .8977). Analysis of the latency of N1 peak amplitude revealed no significant differences. The N1 area analysis revealed a significant effect of Face (F(1, 8) 5 6.22; p , .04; CS1 more negative than CS2). 8

As in many ERP averages associated with subthreshold presentations, distinct component processes (such as N1-P2-P3) are not readily visible in the grand averages. This is due primarily to the nature of brain responses to subthreshold stimuli, and is an area of important future investigation (see Shevrin, 1973, for an early example of this analysis). We conducted this analysis for comparative purposes in order to be consistent with the approach taken in the Precon-Postcon phase analysis.

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FIG. 5. Detailed analysis by stimulus for Pz (a, CS1/unpleasant; b, CS2/pleasant). y axis (at stimulus onset) 5 4 µv increments; x axis 5 400-ms intervals; solid line, preconditioning; dotted line, postconditioning.

The P2 peak amplitude analysis revealed significant main effects of Electrode (F(3, 24) 5 3.20; p , .04; e 5 1.0; F3-F4 . Pz-Cz) and Face (F(1, 8) 5 13.73; p , .006; CS1 less positive than CS2). Analysis of P2 latency revealed a significant effect of Electrode (F(3, 24) 5 7.41; p , .005; F3 . others). The P2 area analysis revealed a significant effect of Face (F(1, 8) 5 13.91; p , .006; CS1 less positive than CS2). Neither P3 amplitude nor area measures were significant, although the means were in a direction consistent with P2. Analysis of P3 latency of peak amplitude revealed a significant Electrode 3 Face interaction (F(3, 24) 5 4.97; p , .009) and a significant effect of Face (F(1, 8) 5 5.99; p , .05; CS1 . CS2), with the interaction the result of a reversal in effect for electrode F3 (CS2 . CS1). On closer inspection of the averages, we observed that the activity in response to presentations of CS1 is generally more negative over time than in response to CS2, from stimulus onset to the shock-noshock event. We decided to assess the significance of the CS1 negativity using area measures that spanned a wider time interval than any specific component area measure.

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FIG. 6. ERP grand average at each electrode: Conditioning phase. y axis (at stimulus onset) 5 2 µv increments; x axis 5 400-ms intervals; arrow, shock-nonshock event at 800 ms; solid line, CS1; dotted line, CS2.

Area Findings We obtained area measures from three regions: (1) Total area (100–700 ms poststimulus), (2) Early area (100–400 ms), and (3) Late area (400–700 ms). Each of the regions was subject to a Face(2) 3 Electrode(4) analysis of variance. For the Total area, there was a marginally significant effect of Face (F(1, 8) 5 4.71; p , .06) in the expected direction (CS1 more negative). No other effects were significant. In the Early area, there was a significant effect of Face (F(1, 8) 5 7.43; p , .03), again, in the expected direction. No other effects were significant. Finally, in the Late area, there were no significant effects though the means for Face were in the expected direction (Face F(1, 8) 5 1.69; n.s.).

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TABLE 2 Conditioning Phase Mean (SD) Values a. Component measure Amplitude (µv)

Pz N1 P2 P3 Cz N1 P2 P3 F4 N1 P2 P3 F3 N1 P2 P3

Latency (mseconds)

CS1

CS2

CS1

CS2

22.7 (1.0) 1.8 (1.4) 3.4 (1.8)

2.9 (1.6) 2.7 (1.9) 4.7 (2.8)

130 (20) 198 (25) 473 (74)

128 (18) 191 (19) 368 (65)

22.8 (1.1) 1.2 (1.3) 3.4 (1.1)

21.2 (2.1) 2.6 (1.0) 4.1 (2.3)

121 (21) 198 (25) 439 (69)

136 (20) 184 (21) 374 (66)

21.4 (1.1) 2.0 (1.2) 4.0 (1.6)

2.9 (1.5) 3.3 (1.2) 4.3 (1.6)

118 (21) 188 (22) 440 (52)

128 (19) 178 (19) 430 (85)

21.6 (2.0) 2.4 (.5) 4.2 (2.0)

21.1 (2.4) 3.5 (1.2) 4.3 (1.6)

118 (17) 186 (20) 422 (113)

120 (19) 168 (10) 436 (99)

b. Area measure

Pz Early area Late area Total area Cz Early area Late area Total area F4 Early area Late area Total area F3 Early area Late area Total area

CS1

CS2

226 (72) 24 (126) 225 (141)

72 (115) 74 (215) 116 (205)

233 (64) 8 (99) 231 (125)

61 (128) 61 (180) 99 (219)

35 (97) 83 (146) 81 (169)

90 (83) 122 (122) 163 (147)

41 (86) 95 (134) 86 (146)

75 (99) 100 (159) 130 (180)

The results from the area analyses indicate that there were significant ERP differences during the Conditioning phase. Specifically, the CS1 became more negative than CS2 shortly after stimulus presentation, and returned to baseline prior to the shock-noshock event. Statistically, this effect was strongest in the Early post-stimulus region (100–400 ms) and decreased in the Late region (400–700 ms). Given that the results of the Conditioning phase are a direct index of the acquisition process, and that the Early area measure is a good indicator of the differences between CS1 and CS2, we conducted an analysis exploring the trial-by-trial development of the Early area difference in this phase. Using trial as a continuous independent variable, we conducted a Face(2) 3 Trial repeated-measures analysis of variance for the Early area. Frontal electrodes yielded no significant effects. For Cz, a Face 3 Trial interaction emerged as a nonsignificant trend in the expected direction (F(1, 8) 5 2.39; p 5 .16); for Pz, a significant trend emerged (F(1, 8) 5 4.70; p 5 .06). As the trials progressed, CS1 became more negative than CS2 in the Early area, especially at Pz. These findings should be understood as a conservative index that underestimates existing differences, given the relatively low sensitivity of single-trial ERPs. Thus, the finding of trends in the expected direction likely indicate actual differences that are much larger and more significant than those reported here.9 9 One could also justify conducting this analysis with a directional hypothesis, specifically that CS1 will become increasingly negative across trials. Such a directional hypothesis would change the criterion for significance to p 5 .1 (Howell, 1987). The findings for Pz then would technically reach a significant level.

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Subjective Ratings Ratings obtained after the experiment revealed that most subjects did not have consistent ideas regarding when the shock would occur. Of the three subjects who ventured hypotheses, one believed that the unpleasant face was linked to the shock and two believed that the pleasant face was linked to the shock. Summary of Conditioning Phase Findings The results of the Conditioning phase analyses provide positive evidence in our third area of inquiry: acquisition of a conditional response to subthreshold stimuli is reflected immediately and directly in scalp recorded brain activity. The component analyses revealed that CS1 is more negative than CS2 for N1 and less positive than CS2 for P2 and P3. The area analyses were quite consistent with the component results. The CS1 became more negative than the CS2 shortly after stimulus presentation, and returned to baseline prior to the shock-noshock event. Furthermore, the CS1 negativity increased across trials, indicating that this negativity indexed a learning process. The CS1 negativity, best reflected in the area measure, is a new finding that supports the experimental hypothesis that acquisition of a conditional response can occur with subthreshold stimuli. DISCUSSION

The nature of nonconscious learning has been a topic of increasing interest in psychology. The cognitive literature indicates that such learning can occur in two distinct ways: in an implicit, knowledge-based way and in a subthreshold, perceptually based way. Implicit learning involves stimuli that are accessible to awareness, but with the rules or algorithms acquired by the individual inaccessible. Subthreshold or subliminal learning, in contrast, involves perceptually inaccessible stimuli. Although the cognitive literature has progressed toward elaborating the nature of nonconscious learning, little work has been accomplished in elucidating the neural systems involved in such processes. The classical conditioning literature that addresses simple associative learning using physiological indices is one area where this topic has generated interest, but the results have been equivocal and at odds with the existing cognitive literature. A major conclusion in the classical conditioning literature is that acquisition of a conditional response cannot occur when stimuli are degraded or rendered inaccessible to awareness (Dawson & Schell, 1985). The present study sought to examine whether nonconscious associative learning (of the perceptual kind) could occur in the context of a classical conditioning paradigm, and whether neural indices of such learning could be measured by scalp recorded electrical activity. Affirmative answers to these questions would provide an important link between the cognitive and neural basis of nonconscious associative learning. The results of this study provide two lines of evidence supporting the conclusion that associative learning can occur outside awareness. First, in the pre- vs postconditioning phase analyses (an indirect measure of acquisition), we discovered differential activity in CS1 and CS2, especially in the predicted P3 component amplitude. And second, in the conditioning phase analyses (a direct measure of acquisition), we dis-

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covered a processing negativity associated with CS1, which appears to index acquisition itself. We now turn to a more extended discussion of these results. Preconditioning vs Postconditioning Phase Findings

The major finding in the suprathreshold pre- vs postconditioning phase analyses was of differential effects for CS1 and CS2. First, as predicted, P3 amplitude for CS1 did not change from preconditioning to postconditioning phase, and was larger than CS2 in the postconditioning phase. This effect is consistent with what has been described as a ‘‘resistance to extinction’’ effect (e.g., Ohman & Soares, 1993). The P3 findings also are consistent with other results using conditioning paradigms. In our previous study, for example, P3 amplitude for CS1 was larger than CS2 in a suprathreshold acquisition series (Wong et al., 1994). Increased component amplitudes (for CS1) as a result of conditioning also were reported by Begleiter and Platz (1969). Though P3 differences in the present study are a consequence of prior subthreshold acquisition of a conditional response (and not a direct index of acquisition), the effects on P3 amplitude are similar. The overall results are consistent with the view that P3 is an index of stimulus salience or emotional value (Begleiter et al., 1983; Johnston et al., 1986; Donchin & Coles, 1988). The second main finding in this analysis is of an overall decrease in amplitude associated with the CS2 in the postconditioning phase. In contrast, the CS1 results demonstrate a ‘‘resistance’’ to the process observed in CS2. These results are consistent with other research findings. Schell et al. (1991), for example, reported that potentially phobic stimuli were more prone to resist extinction than neutral stimuli. It seems likely that certain stimuli, such as faces with expressions of negative affect, may be especially salient (Dimberg, 1986; Esteves et al., 1994a). Supporting this conclusion, Esteves et al. (1994b) recently demonstrated a differential extinction effect in SCR with facial stimuli that had been conditioned outside awareness. This effect was found with angry faces only. One alternative interpretation of the results in the postconditioning phase should be addressed. One could argue that the differences observed are in fact due to intrinsic qualities of the stimuli and not due to conditioning. In other words, differences that emerged later in the postconditioning phase were a function solely of the additional stimulus repetitions, with no effect from the subthreshold acquisition series. While this alternative interpretation (of differential sensitization) is viable in principle, it should be noted that the present study relied on established standards to evaluate stimulus differences in the preconditioning phase. Thus, this interpretation would need to hinge on the premise that the measurement of intrinsic stimulus differences needs more powerful signal-to-noise ratios (i.e., more repetitions) than is ordinarily required. Evidence directly contradicting a differential sensitization hypothesis can be found in the recent work by Esteves et al. (1994b). In two separate experiments, using conceptually identical experimental procedures to the present study, Esteves et al. found no evidence that intrinsic effects of angry and happy faces interacted with conditioning. In Experiment 1, one group included neutral faces as subthreshold CSs, with one face linked to a shock and the other not, during the acquisition or conditioning phase. In the postconditioning extinction phase, a happy and angry face were

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substituted for the (previously neutral) conditional stimuli; no evidence was obtained for a differential SCR response to the happy and angry faces. The only evidence for differential responses to happy and angry faces in a postconditioning extinction phase was when these faces had been used as conditional stimuli in a prior acquisition phase. In Experiment 2, subthreshold angry and happy faces were paired randomly with a US (aversive shock). Again, no evidence was obtained for differential SCR responses to the happy and angry faces when they were later presented in awareness. Both experiments by Esteves et al. (1994b) consistently demonstrate that sensitization effects do not interact with subthreshold conditioning of faces with emotional expressions. In sum, both results—the P3 amplitude advantage for CS1 and the decrease in responsivity for CS2 —provide converging evidence that a conditional response was acquired to subthreshold presentations of the facial schematics. While these findings are an indirect index of the acquisition process, they nonetheless constitute strong converging evidence that a conditional response had been acquired previously to subthreshold presentations of the stimuli. Conditioning Phase Findings

Direct evidence for the acquisition of a conditional response was found in the subthreshold conditioning phase of the experiment. Here, the CS1 elicited greater negativity in the brain potential than did the CS2. This negativity emerged shortly after stimulus presentation and diminished as the shock-noshock event neared. Furthermore, the CS1 negativity increased with each additional trial on a trial-by trial analysis, indicating that the acquisition process was indexed by the CS1 negativity and that subthreshold learning was unfolding over time. These findings are noteworthy, and are highly consistent with the main experimental hypothesis in that differential responses to the CS1 and CS2 were obtained during the acquisition phase itself. The ERP negativity for CS1 involves an initial negative shift, which then gradually diminishes as the shock-noshock event approaches (cf. Early and Late area results). What does this CS1 negativity reflect? We might logically conclude that it reflects some aspect of the acquisition process; however, one could consider two alternative hypotheses. Suppose, for example, the negativity reflects a preexisting difference in the stimuli (a differential sensitization hypothesis), or a consequence of the subthreshold presentation of the stimuli? A number of observations, however, directly contradict these alternative hypotheses. First, a differential sensitization hypothesis would involve the assertion that sensitization occurs in different directions for sub- and suprathreshold stimuli, e.g., CS1 is more positive in the suprathreshold postconditioning phase and more negative in the subthreshold conditioning phase. There is no data in the literature indicating that this pattern exists, and there is no a priori reason to reject the more parsimonious assertion that sensitization effects should be directionally consistent. Second, one way to address the alternative hypotheses would be to present subthreshold affectively valent facial stimuli, without conditioning, to test for intrinsic stimulus differences. This approach, in fact, was taken in our earlier study (Wong et al., 1994). In that study, the same facial stimuli, each presented under virtually identical subthreshold conditions in a baseline, preconditioning phase, elicited no ERP differences between the facial stimuli. Third, a trial-

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by-trial analysis of the Wong et al. (1994) data (which was identical to the analysis conducted in the present study in the Conditioning phase for the Early area) yielded no significant differential effects across time in the baseline phase. Based on these observations, it is unlikely that intrinsic differences in the stimuli emerged in the current subthreshold conditioning phase, nor can it be argued that it is the subthreshold stimulus presentations as such that produce the effect insofar as no such differences were found in a preconditioning baseline subthreshold phase for either component or trial-by-trial analyses. It thus seems likely that the effect observed in the conditioning phase of the present study is a reflection of the acquisition process itself. Additional evidence indicating that the conditioning phase ERP negativity is indexing the acquisition process comes from concurrent measures of facial EMG responsivity using the same paradigm (Bunce, 1996; Bunce et al., 1997). For facial EMG, Bunce et al. demonstrated a trial-by-trial increase in level of activity across the conditioning phase for CS1, while CS2 showed no such increase. This trial-bytrial analysis, which is an even more robust analysis with facial EMG activity due to higher sensitivity and signal-to-noise ratios than ERPs, is yet another indication that subthreshold learning is unfolding during the conditioning phase. Although the exact correspondence between the facial EMG activity and the ERP negativity is still unknown (and in need of further investigation), it is clear that the facial EMG activity is differentiating CS1 and CS2 in ways that are consistent with what was discovered in the present study using ERPs. Each measure appears to be indexing, via different psychophysiological ‘‘windows,’’ acquisition of a conditional response to the subthreshold facial stimuli. In structure, the conditioning phase ERP negativity is similar to the expectancy wave (e-wave) observed in the Wong et al. (1994) study. This e-wave was obtained in response to subthreshold presentations of faces previously conditioned in awareness. The results of the present study, however, derive from a slightly different paradigm; with an 800-ms ISI, we were not expecting an e-wave because previous research highlighted the importance of longer ISIs in the development of an expectancy process (e.g., Backs & Grings, 1985). Thus the negativity discovered in this study, although perhaps related to an expectancy process, also may reflect something different. What exactly is different, however, is an open question. For example, might this early negativity be related to what others have identified in the auditory sphere as a ‘‘processing negativity’’ (Naatanen, 1990), or with an anticipatory motor response partially reflected in facial EMG (Bunce et al., 1997)? The negativity also may reflect face-specific processing. Hallgren (1992), for example, reports widespread negativity at 225 ms in response to faces; this negativity attains maximal amplitude in the amygdala in depth recordings. If the conditioning phase negativity reflects face-specific neuronal responses that involve activity in the amygdala, then a neural basis for subthreshold stimulus responsivity may be at hand. In view of the potential role of the amygdala in the conditioning phase negativity, and its importance in processing affectively valent stimuli, one aspect of the study should be explored further. We assume (based on well-established findings, e.g., Esteves et al., 1994) that the affective valence of the CS, especially the unpleasant face, is particularly salient regarding conditioning. In making such an assumption, we were not exploring affective valence per se in the acquisition process (and it is

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not logically necessary to do so in order to establish that a conditional response can be acquired). However, given the potential relevance of affective valence of the CSs on the conditioning process, further exploration of this issue is warranted. For example, one might undertake a replication of the present study using a full factorial design during acquisition (i.e., where both pleasant and unpleasant faces serve as CS1), which would address directly whether subthreshold acquisition requires a negatively valent face or whether faces of either valence would work as well. Based on the work of Esteves et al. (1994) where a full-factorial design was used with SCR, one might anticipate that subthreshold conditioning effects will be observed only with unpleasant faces. Other questions pertaining to the nature of the CSs also can be addressed, such as whether unconscious associations can be established to affectively valent stimuli other than faces, or whether affectively valent stimuli are needed at all. Overall, the ERP negativity in the conditioning phase seems to index brain processes associated with the nonconscious acquisition of a conditional response to faces. The negativity may reflect an expectancy or anticipatory process that emerges once acquisition has occurred, some other yet to be identified process related to stimulus selection outside awareness, or a combination of the two. The negativity also may provide us with neural evidence for subthreshold face-specific processing. Importantly, all of these processes occur entirely outside awareness. Implications for Conscious and Nonconscious Processes

In this study, we provide two lines of evidence supporting the conclusion that associative learning can occur outside awareness as reflected in brain activity. First, in the postconditioning phase we find indirect evidence of acquisition reflected in the differential activity of the suprathreshold CS1 and CS2. And second, we find direct evidence of acquisition in the conditioning phase, reflected in a processing negativity associated with the subthreshold CS1 and not CS2. Of note is that this negativity increases with additional trials, indicating that a learning process is unfolding during the conditioning phase. Thus, we have converging evidence, both direct and indirect, indicating that acquisition of a conditional response can occur with stimuli that are inaccessible to awareness. Other studies (Ohman & Soares, 1993; Wong et al., 1994) have demonstrated that a previously acquired conditional response can be elicited at a later time by subthreshold presentations of the stimuli. The present study extends these results into the area of nonconscious associative learning, and provides evidence that learning can occur with stimuli rendered perceptually inaccessible to awareness. The combined evidence from these recent conditioning studies indicates that mental processes associated with acquisition, expectancy and extinction can be elicited by perceptually inaccessible stimuli. Nonconscious processes, as indexed by these paradigms, appear to be somewhat more complex than others have described (Greenwald, 1992). The present results also are consistent with other experiments demonstrating nonconscious learning (e.g., Kunst-Wilson & Zajonc, 1980; Esteves et al., 1994), and extend these processes into the physiological domain as reflected in brain activity. The present study raises several interesting questions regarding the nature of nonconscious learning and its effect on conscious processes. Based on the CS1/CS2

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results in the suprathreshold postconditioning phase, it seems that what is learned outside awareness can have an effect on subsequent stimulus presentations that are fully accessible to awareness (i.e., when subjects can see the faces). It’s not evident, however, whether any subjective experience, other than a perceptual experience, was elicited in subjects in the postconditioning phase. For example, in post-experiment questionnaires and interview data, there was no indication of alteration in subjective experience. Subjects reported no reactions to the stimuli in the postconditioning phase that were different from what was experienced in the preconditioning phase. This suggests that differential responses can occur with perceptually accessible stimuli without the person being aware, in a knowledge-based sense, of the processes themselves. Similarly, post-experiment ratings of the subthreshold conditioning phase indicated that even though subjects acquired a differential response to perceptually inaccessible stimuli, there were no consistent consciously articulated knowledgebased ideas regarding why or when the shock would occur. The distinction we are making between perceptual awareness and knowledgebased awareness is important in several regards.10 The research we have described in this and in our previous report (Wong et al., 1994) suggests that mental processes in the context of a conditioning paradigm can be elicited when stimuli are perceptually inaccessible. The effects of these nonconscious processes on conscious, knowledgebased processes are not yet clear. It seems likely, however, that knowledge-based processes are only partially correlated with an individual’s responses to both perceptually accessible and inaccessible stimuli. Additional exploration is needed of the interface between conscious processes, in both the perceptual and knowledge-based sense and the nonconscious processes. Furthermore, conditioning paradigms such as the one used in the present experiment, in which stimuli in various stages of the conditioning process are perceptually inaccessible, seem well suited to the investigation of nonconscious learning. REFERENCES Backs, R. W., & Grings, W. W. (1985). Effects of UCS probability on the contingent negative variation and electrodermal response during long ISI conditioning. Psychophysiology, 22, 268–275. Barnett, V., & Lewis, T. (1984). Outliers in statistical data. New York: Wiley. Begleiter, H., & Platz, A. (1969). Evoked potentials: Modifications by classical conditioning. Science, 166, 769–771. Begleiter, H., Porjesz, B., Chou, C. L., & Aunon, J. I. (1983). P3 and stimulus incentive value. Psychophysiology, 20, 95–101. Bower, G. H. (1990). Awareness, the unconscious, and repression: An experimental psychologist’s perspective. In J. L. Singer (Ed.), Repression and dissociation. Chicago: University of Chicago Press. Brandeis, D., & Lehmann, D. (1986). Event-related potentials of the brain and cognitive processes: Approaches and applications. Neuropsychologia, 24, 151–166. Bunce, S. C. (1996). Facial EMG and ERP indices of emotion-relevant unconscious learning. Paper presented at the annual meeting of the American Psychological Association, Toronto. Bunce, S. C., Bernat, E., Wong, P. S., & Shevrin, H. (1997). Evidence for unconscious learning: Facial EMG indicators of unconscious associative learning. Under review.

10

Note that other researchers have made similar distinctions, e.g., Bower (1990) and Hirst (1995).

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