Differential conditioning of anticipatory pupillary dilation responses in humans

Biological Psychology 60 (2002) 51 – 68 www.elsevier.com/locate/biopsycho

Differential conditioning of anticipatory pupillary dilation responses in humans Gu¨nter Reinhard *, Harald Lachnit * Department of Psychology, Philipps-Uni6ersita¨t Marburg, Gutenbergstr. 18, D-35032 Marburg, Germany Received 23 March 2001; accepted 16 January 2002

Abstract The purpose of the present experiments was to demonstrate conditioning of anticipatory pupillary dilation responses in humans as previous efforts to condition pupillary dilation had yielded inconsistent results. In those studies, using an aversive unconditioned stimulus (US) had yielded more promising results than using non-aversive USs. In three experiments we utilized a reaction time task as a non-aversive US and observed reliable differential conditioning of anticipatory pupillary dilation. Differential responding was evident within very few trials and was largest late in the anticipatory interval. Experiment 4 replicated these findings using an aversive US (electric shock). In measuring anticipatory responses in a differential conditioning preparation all four experiments provided clear evidence for Pavlovian conditioning of pupillary dilation in humans. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Pavlovian conditioning; Pupillary response; Humans

1. Introduction The purpose of the present experiments was to demonstrate differential Pavlovian conditioning of pupillary dilation responses in humans in order to broaden our arsenal of methods for the examination of the processing of compound stimuli. Previous studies of human Pavlovian conditioning that utilized autonomic re* Corresponding author. Tel.: + 49-6421-2823438; fax: +49-6421-2826621. E-mail addresses: [email protected] (G. Reinhard); [email protected] (H. Lachnit). 0301-0511/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 5 1 1 ( 0 2 ) 0 0 0 1 1 - X

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sponses, heart rate or mostly skin conductance responses (SCR), have provided less convincing data in reference to characteristics of Pavlovian conditioning such as gradual acquisition functions or lack of complete habituation over trials than have studies of eyelid conditioning: (1) neutral stimuli, in particular the first presentation of the conditioned stimuli (CSs), led to responding; (2) the acquisition function was not negatively accelerating and acquisition occurred within the first few trials; and (3) all responses, including the unconditioned responses, habituated over trials. For a cognitive approach to Pavlovian conditioning (e.g. Rescorla, 1988), however, these shortcomings are not essential to the extent that responding changes with changing relations between stimuli. The main focus of interest within this approach is on the relations (associations) between representations of environmental events. In this view, the conditioned response is only used as an indicator of associations. For example, when a CS is followed by an unconditioned stimulus (US) and the interstimulus interval (time from CS onset to US onset) is sufficiently long (e.g. 8 s), an enhanced anticipatory SCR can be observed on subsequent trials. To the extent that a CS signaling a US (CS+ ) leads to larger SCRs than a CS signaling no US (CS−), anticipatory SCRs may serve as an indicator of outcome expectancy. Furedy and Riley (1987), too, discussed this empirical criterion (CS + \ CS −) for conditioning in ‘defending’ the CS-elicited SCR from being dismissed as a non-conditioned response. Previously, we have used differential Pavlovian anticipatory SCR conditioning to evaluate whether stimulus compounds are processed as the sum of their elements or as unique configurations (for a recent overview of this old debate, see Pearce and Bouton, 2001; Wasserman and Miller, 1997). In five series of experiments (Lachnit and Kimmel, 1993, 2000; Lachnit and Lober, 2001; Lachnit et al., 2001, 2000) we have reported converging evidence that humans utilize rules in order to solve certain discrimination problems in differential Pavlovian conditioning. Although the elements have to be processed in applying these rules, applications of the rules lead to ‘configural like’ observations. This utilization of rules could be observed empirically, because it leads to expectancies about potential outcomes following certain CSs. These expectancies are mirrored by anticipatory SCRs. In a recent report (Reinhard and Lachnit, 2002), we have shown that pupillary dilation can be used as a valid indicator of cognitive processing during human learning and during categorization in a Go/NoGo task. Experiment 1 provided evidence that expectancies developing in the course of categorical rule learning can override stimulus driven processing and Experiment 2 successfully explored compound processing. Hence, we decided to utilize anticipatory pupillary dilation responses in a differential conditioning preparation. Although Pavlovian conditioning of pupillary responses is well established in cats and dogs (e.g. Cassady, 1996; Harlow and Stagner, 1933; for an overview see Lennartz and Weinberger, 1992), previous attempts to condition the pupillary response in humans failed to obtain reliable results (for an historical overview of earlier work see Voigt, 1968). Taking into account the inconsistent findings for pupillary constriction, Young (1965) concluded that conditioning of pupillary constriction cannot occur. Until now

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there is no good reason to reject this conclusion. The empirical data are also inconsistent with regard to conditioning of pupillary dilation (e.g. Fitzgerald and Brackbill, 1968; Tanck, 1970). It seems that only Gerall (Gerall et al., 1957; Gerall and Woodward, 1958) provided convincing evidence for conditioning of pupillary dilation in humans using shock instead of light as the US. Considering the pupillary dilation as part of a generalized unconditioned response to noxious stimuli, Young (1965) concluded that the only evidence for conditioning concerns the dilation of the pupil to a noxious stimulus. Kugelmass et al. (1969) reported supporting results using an aversive auditory stimulus as the US. In brief, since the pioneer work of Watson (1916) there is only weak evidence for pupillary conditioning in humans. Furthermore, there is no convincing empirical evidence for conditioning of pupillary dilation with a non-aversive US. Thus, one main purpose of the present study was to demonstrate conditioning as indexed by pupillary dilation in humans with a non-aversive US. We used a differential conditioning paradigm mainly because all of our conditioning studies employed this preparation, which controls for alpha responses and pseudo conditioning. Furthermore, we used interstimulus intervals long enough to allow for the measurement of anticipatory responses in the course of training without any need for extra test trials. Richer (Richer and Beatty, 1985; Richer et al., 1983, Experiment 1) demonstrated that not only movement execution but also preparation for a movement evokes pupillary dilations. This raises the question as to whether it is possible to assess these movement-related (anticipatory) pupillary responses within a differential conditioning paradigm. Richer et al. (1983, Experiment 2) described a Go/NoGo task with a delayed response condition. Participants were told to withhold a motor response after presentation of a tone until a second tone was presented. When examining the averaged pupillary responses immediately before the second tone, the pupil diameter on Go trials was nearly 0.25 mm larger than on NoGo trials, a consequence of dilation. The first tone in this procedure served similar purposes as do the CSs in differential conditioning, hence we reasoned that it should be possible to use a reaction time (RT) task as the US in conditioning pupil dilation. This proposal was supported by the fact that an RT task had already successfully been used as the US in Pavlovian conditioning. Marquis and Porter (1939) found that an RT task was an effective US in eyelid conditioning. Moreover, an RT procedure was used in conditioning of the heart rate (e.g. Lipp and Vaitl, 1988) and digital pulse volume responses (e.g. Lipp et al., 1992). Furthermore, there are studies in which an RT task led to electrodermal conditioning (e.g. Baer and Fuhrer, 1969; Fuhrer and Baer, 1970; Lipp et al., 1994; Lipp and Vaitl, 1992; Siddle et al., 1985). In addition, it is possible to observe conditioned motor responses (e.g. key pressing) when using an RT task (Montare, 1992). Lipp and Vaitl (1990) described a procedure for differential conditioning with an RT task as the US. A non-aversive tone was used as the imperative stimulus and participants were asked to press a button as fast as possible after hearing the tone. In acquisition, a visual stimulus was paired with the tone (CS+ ), whereas another was presented alone (CS− ). The tone coincided with CS+ offset and was terminated by the participant’s response. This procedure resulted in reliable differ-

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ential conditioning of heart rate, digital pulse volume and electrodermal responses. Therefore, it was regarded as a promising tool for differential conditioning of anticipatory pupillary dilations in humans. Furthermore, this procedure permits comparisons with findings from studies of anticipatory SCR conditioning, the most frequently used paradigm in the study of human Pavlovian conditioning.

2. Experiment 1 Similar to Lipp and Vaitl (1990), we used two visual stimuli, letters, as CSs in Experiment 1. Each of the two CSs was presented for 8 s. The CS+ was immediately followed by a tone, which had to be responded to by a finger movement.

2.1. Method 2.1.1. Participants Fifteen University of Marburg students participated in Experiment 1. They received either course credit or were paid for participation. The data of 3 participants were excluded from the analyses because of too many eye blinks during the measurement of pupillary responses. Hence, the analyses were conducted on data from 12 participants, 6 females and 6 males. Their mean age was 24.8 years (range, 20 –29). 2.1.2. Apparatus and stimulus material Apparatus, calibration procedures and stimulus material were similar in all experiments reported here. The size of the right horizontal pupil diameter was measured by an infrared light eye camera (IVIEW system for Windows from SensoMotoric Instruments GmbH), that was attached to the participant’s head. A computer sampled pupillary responses at 20 Hz with an accuracy of 0.027 mm (SD =0.003 mm). The experimenter calibrated the system by placing a black dot of a specific size on the closed lid of the right eye. With this procedure, conducted at the beginning and at the end of the experimental session, the system was able to track the actual diameter of the pupil throughout the experiment. Testing took place in a sound-attenuated room. The participant was seated in a chair facing a computer screen located 1.5 m away. The screen was enclosed within gray painted wooden panels, forming a funnel-shaped device resembling a megaphone, 90 cm in length. The participant sat in front of the wide opening (100× 100 cm2) of the funnel, and the screen containing the stimuli was placed directly behind the narrow opening (22×13 cm2). The room lights were turned off and only the inside of the funnel was illuminated, leading to an average pupil diameter of about 3.9 mm. We used visual stimuli as CSs. Black lines (1 mm wide) on a green background forming two letters (C and N; each 3 cm wide, 4.2 cm high) were used as CS+ or CS − , respectively. The letter C was always presented 8 mm to the left of the center

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and N was always presented 8 mm to the right. Measurements with a light meter showed that presentation of the letters did not produce any appreciable change in luminance. Such ‘non-pictorial stimuli’ may be used during measurement of the pupil (see e.g. Janisse, 1977). Furthermore, we successfully used such letters when exploring the influence of stimulus probability on pupillary dilation (Reinhard and Lachnit, 2002). The imperative stimulus of the RT task was a non-aversive tone of 2500 Hz (60 dB(A)).

2.1.3. Procedure The participants were informed that the purpose of the experiment was to measure their physiological responses to various stimuli (letters on a screen as well as occasional tones), and that they should try to avoid unnecessary movement, speaking and heavy breathing. They were asked to focus on a fixation cross in the middle of the display. Participants were instructed to start a trial by pressing a key with the index finger of the preferred hand when the message ‘Press the key!’ appeared on the display. They were informed that their task was to pay close attention to the letters presented and to release the key as fast as possible when they heard a tone. Participants were also instructed to avoid errors. A letter was presented for 8 s, 3.5 s (91 s) after key pressing. The tone lasted for a maximum of 5 s if it was not terminated by the participant’s response. Finally, 5 s after CS offset, the message ‘Release the key!’ signaled the end of the pupil measurement. The participants were not informed about the contingencies between the CSs and the tone. On paired conditioning trials, the tone coincided with CS+ offset. The interstimulus interval (CS onset to tone onset) was 8 s. The intertrial interval (CS onset to CS onset) equaled 2493 s (in some cases it was longer because it was up to the participant to start the trial). Twenty-four trials of CS+ followed by the tone and 24 trials of CS− without the tone were presented in random order with the restriction that blocks of 8 trials each contained 4 trials of CS+ and 4 trials of CS −, and that no more than three reinforced or non-reinforced CSs occurred consecutively. The experiment lasted for about 25 min. At the end of the experimental session, the second pupillary calibration was performed. 2.1.4. Dependent 6ariable The individual records were inspected and small artifacts (e.g. eye blinks) were corrected by linear interpolation. Trials containing more serious artifacts (mostly about 500 ms or more in duration) affecting critical intervals of the trial (500 ms before CS onset until CS offset) were eliminated (2.6% of the trials). The initial pupil diameter was determined for every artifact-free record as the average pre-CS diameter across 200 ms. Also, mean pupillary sizes were determined during four different time intervals, 0– 2, 2– 4, 4–6, and 6–8 s after CS onset. The differences between these values and the initial diameter were calculated and averaged separately for reinforced and non-reinforced trials into four blocks of 6 trials each. Because some trials had to be eliminated, some blocks contained only 5 (6.25% of the blocks) or fewer than 5 trials (3.13% of the blocks).

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2.2. Results and discussion The significance level was set to P B 0.05 in all statistical tests across all experiments, and stated probability levels are based on the Huynh and Feldt (1976) adjustment where appropriate. Fig. 1 depicts pupillary alterations starting from CS onset for 9 s, averaged across participants separately for CS+ and CS − in four blocks of 6 trials each. The large dilation after the offset of CS+ is typical for an index finger response in an RT task (e.g. Richer and Beatty, 1987). As can be seen, already in Block 1 pupillary dilations during CS+ were larger than during CS− . Overall, this difference seemed to be largest during the latest time interval. These impressions were supported by the analysis of variance (ANOVA). A 2× 4 × 4 (Contingency× Block × Interval) ANOVA yielded a main effect for contingency that indicated response differentiation, F(1, 11)= 8.11, MSE= 0.0486, PB 0.02. Response differentiation increased linearly across intervals, F(1, 11)= 7.76, MSE= 0.0126, PB 0.02. There was neither a variation of response differentiation across blocks, F B1, nor was the three-way-interaction significant, F(9, 99)=1.57, MSE = 0.0036, P \ 0.16.

Fig. 1. Average pupillary responses during CS + and CS− in Experiment 1 as a function of blocks of 6 trials. The vertical lines mark the limits of the relevant time windows. The arrow indicates CS offset.

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Fig. 2. Average pupillary responses during the first and second trials of CS + and CS − in Experiment 1. The vertical lines mark the limits of the relevant time window. The arrow indicates CS offset.

In a second step we computed 2× 4 (Contingency× Block) ANOVAs for each interval separately. Differences in pupillary dilation between CS+ and CS− increased continuously from the first to the fourth interval (F values of the main effects for contingency were 2.06, 4.09, 7.57, and 10.07; the largest F for the Contingency×Block interaction was 1.25). Fig. 2 shows pupillary alterations during the first and during the second trials of acquisition only. The figure illustrates that the response differentiation between CS + and CS − developed very early in training. Although an analysis of single trials is not very reliable due to spontaneous pupil fluctuations, and although the data of one participant had to be excluded from the analysis due to artifacts, we computed a 2× 2 (Contingency × Trial) ANOVA for the fourth interval. The results suggested that response differentiation, as indicated by a main effect for contingency, tended to be evident very early, F(1, 10)=3.72, MSE= 0.0602, P = 0.083, and to increase from the first to the second trial, Contingency×Trial interaction, F(1, 10)=4.43, MSE= 0.0315, P= 0.062. Taken together, Experiment 1 provided evidence for differential conditioning of anticipatory pupillary dilation in humans. There was no difference between CS+ and CS− at the very beginning of training, the differentiation developed very early and was found throughout acquisition. Consistent with the findings of Richer et al. (1983), response differentiation was most pronounced late in the trial. Contrary to previous research, we found conditioning of pupillary dilation with a non-aversive US by using a differential anticipatory conditioning preparation.

3. Experiment 2 Experiment 1 showed that differential conditioning of anticipatory pupillary dilation in humans can be obtained with a non-aversive RT task as the US. The interstimulus interval (8 s) was the same as used by Lipp and Vaitl (1990). It remains unclear, however, whether or not such a long interval is optimal for

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conditioning of anticipatory pupillary dilation. Gerall and Woodward (1958) found that conditioning was optimal with an interval of 1.5 s, marginal with a 0.5 s as well as with a 0.125 s interval, and not evident with a 2.5 s interval. Richer et al. (1983) used an interstimulus interval (first to second tone) of 3 s. Their intertrial interval was likely to be shorter as that in Experiment 1, but an exact specification of the intertrial interval is not possible because participants indicated verbally that a trial could proceed. Therefore, it may well be that differential anticipatory conditioning could be stronger when shorter interstimulus and intertrial intervals are used. Experiment 2 was conducted to investigate this hypothesis. A further option to enhance anticipatory conditioning could be to increase the significance of the RT task or to enhance the involvement of the participants. Lipp and Vaitl (1988, 1990) demonstrated that positive feedback enhanced SCR conditioning with an RT task as the US: graded feedback (‘good’, ‘very good’ or ‘excellent’) was given whenever the RT was faster than the mean RT during previous trials. We decided to enhance the involvement of our participants by informing them at the beginning of Experiment 2 that the participant with the shortest response time would win a certain amount of money.

3.1. Method 3.1.1. Participants Twelve University of Marburg students (6 females and 6 males) served as participants in Experiment 2. Their mean age was 22.7 years (range, 19–28). None of them had taken part in Experiment 1. They received either course credit or were paid for participation. 3.1.2. Apparatus and stimulus material Apparatus, calibration procedures and stimulus material were the same as in Experiment 1. 3.1.3. Procedure The procedure was very similar to that of Experiment 1 with the following main differences. The participants were informed that they would receive 15 Deutsche Mark, if they produced the top performance (minimum of errors and of RT). The participants were also instructed to avoid blinking during the measurement of pupil size. This was done to reduce the number of artifacts. The interstimulus interval was reduced to 4 s. The intertrial interval equaled 159 2 s (in some cases it was longer because it was up to the participant to start the trial). The experiment lasted for about 15 min. 3.1.4. Dependent 6ariable The individual records were inspected as in Experiment 1 (0.87% of the trials had to be eliminated). The initial diameter was determined as in Experiment 1. Mean pupillary sizes during two different time intervals after CS onset were calculated: 0 – 2 and 2–4 s after CS onset. The differences between these values and the initial

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diameter were averaged and blocked as in Experiment 1. Because some trials were eliminated, some blocks contained only 5 (3.13% of the blocks) or 4 trials (1.04% of the blocks).

3.2. Results and discussion Fig. 3 depicts pupillary alterations starting from CS onset for 5 s, averaged across participants separately for CS+ and CS − in four blocks of 6 trials each. As in Experiment 1, pupillary dilations during CS+ were larger than during CS− already in Block 1. Again, this difference seemed to be largest immediately before CS offset. A 2× 4 ×2 (Contingency ×Block × Interval) ANOVA yielded no significant main effect for contingency, F(1, 11)=3.48, MSE= 0.0293, P\ 0.08. Response differentiation tended to vary across intervals, F(1, 11)= 4.86, MSE =0.0091, P = 0.05. Again, there was no variation of response differentiation across blocks, and no significant three-way-interaction, both FsB 1. A 2×4 (Contingency ×Block) ANOVA was performed for the late interval separately. Response differentiation was significant, F(1, 11)= 4.95, MSE= 0.0284, PB 0.05, whereas the Contingency× Block interaction was not, FB 1.

Fig. 3. Average pupillary responses during CS + and CS− in Experiment 2 as a function of blocks of 6 trials. The vertical lines mark the limits of the relevant time windows. The arrow indicates CS offset.

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Fig. 4. Average pupillary responses during the first and second trials of CS + and CS − in Experiment 2. The vertical lines mark the limits of the relevant time window. The arrow indicates CS offset.

As in Experiment 1, the response differentiation between CS+ and CS − developed very early in training. This is illustrated in Fig. 4, showing pupillary alterations during the first and second trial only. However, the corresponding 2× 2 (Contingency× Trial) ANOVA for the late interval yielded neither a significant main effect for contingency, F B1, nor a significant Contingency× Trial interaction, F(1, 11)= 1.82, MSE= 0.0251, P \ 0.2. Thus, in Experiment 2 we again found evidence for differential conditioning of anticipatory pupillary dilation in the interval immediately before the onset of the RT task. Contrary to Experiment 1, where response differentiation early in training tended to be evident, in Experiment 2 we found not a hint of a statistical significant response differentiation within the first two trials. The manipulation of the instructions and of the interstimulus and intertrial interval did not enhance differential pupillary dilation conditioning. This was supported by a cross-experimental 2× 4×2 (Contingency×Block × Experiment) ANOVA for the last time interval. Neither the Contingency×Experiment interaction nor the three-way-interaction were significant, both Fs B1. Although there is the problem of proving the null hypothesis, an interstimulus interval of 4 s did not seem to result in more pronounced conditioning than did an interval of 8 s.

4. Experiment 3 Whereas Experiment 2 differed from Experiment 1 with respect to both instructions and interstimulus (and intertrial) intervals, Experiment 3 was conducted to explore the specific influence of enhanced task involvement on differential anticipatory conditioning. Therefore, as in Experiment 2, we attempted to enhance the involvement of our participants, but now we used the same interstimulus interval (8 s) as in Experiment 1.

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4.1. Method 4.1.1. Participants Twelve University of Marburg students (11 females, 1 male) with a mean age of 21.3 years (range, 19– 25) served as participants in Experiment 3. None of them had taken part in Experiments 1 or 2. They received either course credit or were paid for participation. 4.1.2. Apparatus and stimulus material Apparatus, calibration procedures and stimulus material were the same as in Experiment 1. 4.1.3. Procedure The procedure was the same as in Experiment 1 with two exceptions. First, as in Experiment 2, participants were instructed that the best performance (minimum of errors and of RT) would be rewarded with 15 Deutsche Mark. Second, as in Experiment 2, participants were also instructed to avoid blinking during the measurement of pupil size. 4.1.4. Dependent 6ariable Individual records were inspected (0.69% of the trials had to be eliminated) and the initial diameter was determined as in Experiments 1 and 2. Because Experiments 1 and 2 had shown reliable response differentiation in the interval immediately before CS offset, only the mean pupillary size during the last two seconds of the CS presentation was determined in Experiment 3. The difference between these values and the initial diameter was averaged and blocked as in Experiment 1. Because some trials had to be eliminated, some blocks contained only 5 trials (4.17% of the blocks). 4.2. Results and discussion Fig. 5 depicts pupillary alterations starting from CS onset for 9 s, averaged across participants separately for CS+ and CS− in four blocks of 6 trials each. As can be seen, responses did not differ at Block 1, and response differentiation increased from Block 1 to Block 4. A 2×4 (Contingency ×Block) ANOVA yielded a main effect for contingency, F(1, 11)=7.01, MSE=0.0455, P B0.03, and no significant Contingency×Block interaction, F(3, 33) =2.35, MSE=0.0221, P\ 0.09. In order to evaluate the impact of involvement on response differentiation we conducted a cross-experimental 2× 4×2 (Contingency ×Block ×Experiment) ANOVA for the late time interval in Experiments 1 and 3. We did not include Experiment 2 into this comparison in order to avoid confounding by differences in interstimulus intervals. Response

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differentiation in Experiment 3 did not differ from that in Experiment 1 (Contingency× Experiment interaction: F B 1) and there was no significant three-way-interaction, F(3, 66)= 1.3, MSE=0.0202, P \ 0.28. Thus, increasing the personal involvement of the participants by the instructions did not lead to larger response differentiation (again one might argue that there is a problem of proving the null hypothesis).

5. Experiment 4 Experiments 1, 2, and 3 demonstrated reliable differential conditioning of anticipatory pupillary dilation with a non-aversive RT task as the US, although previous research —if at all—had found evidence for conditioning of pupillary dilation using aversive USs only. In Experiment 4, we used an aversive electric shock US for two reasons. First, we wanted to generalize our findings to aversive USs, too. Second, we attempted to keep in contact with differential SCR conditioning studies of our group.

Fig. 5. Average pupillary responses during CS + and CS− in Experiment 3 as a function of blocks of 6 trials. The vertical lines mark the limits of the relevant time window. The arrow indicates CS offset.

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5.1. Method 5.1.1. Participants Thirteen University of Marburg students served as participants in Experiment 4. None of them had taken part in the previous experiments. They received either course credit or were paid for participation. The data of one participant were excluded from the analyses because of too many eye blinks during the measurement of pupillary responses. Thus, the analyses were performed on data from 12 participants, 8 females and 4 males. Their mean age was 22.1 years (range, 20–26). 5.1.2. Apparatus and stimulus material Apparatus, calibration procedures and stimulus material were the same as described in Experiments 1– 3. In contrast to these experiments, however, a DC electric shock served as the US. The shock was delivered via Ag/AgCl electrodes to the volar surface of the participant’s arm and produced by an isolated transformer– condensor shock generator (Kimmel et al., 1980). The participants adjusted the intensity of the shock individually so that it would be ‘definitely unpleasant but not really painful’. Shock duration was 10 ms. In Experiment 4, no tone was presented and no finger movement was required. 5.1.3. Procedure The procedure was very similar to that of Experiment 3 and only main differences from Experiment 3 will be described. The shock electrodes were attached before the camera was fixed at the participant’s head. The participants were informed that letters would be presented on the screen as well as occasional electric shocks. The contingencies, however, were not mentioned. In contrast to the other experiments, no RT task was presented, instead offset of CS+ coincided with the presentation of the shock US. 5.1.4. Dependent 6ariable The individual records were inspected as in Experiments 1–3 (1.39% of the trials were eliminated). The changes in pupil diameter were defined in four intervals and blocked as in Experiment 1. Because some trials had to be eliminated, some blocks contained only 5 trials (8.33% of the blocks). 5.2. Results and discussion Fig. 6 depicts pupillary alterations starting from CS onset for 9 s, averaged across participants separately for CS+ and CS − in four blocks of 6 trials each. The large dilation after the offset of CS+ was caused by the electric shock. Pupillary dilations during CS+ were larger than during CS− beginning from Block 2. A 2× 4×4 (Contingency × Block ×Interval) ANOVA yielded a main effect for Contingency, F(1, 11)=8.53, MSE=0.0626, PB 0.02. Response differentiation, however, did not vary across intervals or blocks, and there was no significant three-way-interaction, all Fs B 1.

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Fig. 6. Average pupillary responses during CS + and CS− in Experiment 4 as a function of blocks of 6 trials. The vertical lines mark the limits of the relevant time windows. The arrow indicates CS offset.

Experiment 4 demonstrated that differential conditioning of anticipatory pupillary dilation is also found with shock as the US. In contrast to the experiments with an RT task as the US, however, response differentiation was about equally strong throughout all four intervals.

6. General discussion The main purpose of the current study was to demonstrate reliable differential conditioning of anticipatory pupillary dilation in humans. Experiment 1 provided clear evidence that a non-aversive RT task is an effective US for conditioning of anticipatory pupillary dilation. Experiments 2 and 3 replicated this finding. The experiments showed that, when using an RT task as the US, measurement of response differentiation late in the trial provides reliable evidence for conditioning. Furthermore, Experiment 2 showed that shortening the interstimulus interval from 8 to 4 s did not increase response differentiation. This is in accordance with the finding of Jennings et al. (1998) that in a simple RT task pupillary dilation is larger prior to the imperative stimulus during longer foreperiods. Also, as indicated by

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Experiment 3, enhancing the personal involvement of the participants did not improve conditioning. Finally, the outcome of Experiment 4 suggested that electric shock also is an effective US for conditioning of anticipatory pupillary dilation in humans. In contrast to the use of an RT task as the US, however, response differentiation was about the same across all four measurement intervals. Taken together, measuring anticipatory pupillary dilation as the conditioned response in differential conditioning procedures may well serve as an index of associations between representations of environmental events (predictors and outcomes). Although in the previous literature there is only weak evidence for conditioning of pupillary dilation in humans with an aversive US and no convincing empirical evidence for conditioning of pupillary dilation with a non-aversive US, all experiments reported in this study produced reliable results. We attribute our success in demonstrating conditioning in this response system to two facts. In contrast to nearly all earlier attempts, we increased the sensitivity for detecting conditioning by using a differential conditioning paradigm. Even more importantly, we utilized the pupil’s capacity to index stimulus expectancy by measuring anticipatory responses prior to the presentation of the US rather than assessing conditioned responses on US omission test trials in a time window where a US was presented previously. Pupillary dilation may have some advantages in comparison to other indices of autonomic responding and may therefore supplement SCR conditioning in research on human associative learning. The pupillary dilation is used in very different ways in the exploration of cognitive processes. For example, pupillary dilation measurement was extended to the study of short-term as well as long-term memory (e.g. Peavler, 1974), of language processing (e.g. Hyo¨ na¨ et al., 1995), and of attention (e.g. Beatty, 1982). Kahneman et al. (1969) measured pupillary dilation, heart rate, and skin resistance during mental tasks. They reported similar changes in all three psychophysiological systems, but the responses of the pupillary system were most reliable. This is in accordance with the observation of high statistical reliability even with the very small samples commonly employed in pupillometric research (Beatty and Lucero-Wagoner, 2000). Furthermore, in reference to the work of Just and Carpenter (Just and Carpenter, 1993; Just et al., 1996), Beatty and Lucero-Wagoner (2000) pointed out that there is an interesting relationship between pupillary dilation and brain activity. For example, some authors (e.g. Chapman et al., 1999; Friedman et al., 1973) reported strong parallels between pupillometric indices and brain evoked potential components such as P300. This does not mean, however, that pupillary dilation and P300 index the same processes (for an overview of parallels and differences between pupil and P300 see Steinhauer and Hakerem, 1992). In addition, pupillary dilation is often measured during RT tasks (e.g. Richer et al., 1983), because it provides a continuous index of phasic changes in processing demands across the duration of the task. In this regard, it circumvents the problems associated with probe RT or other secondary tasks as indices of primary task processing. The measurement of pupillary dilation during RT tasks is advantageous because the pupil is a relatively fast response system. For instance, the human pupil

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responds to light stimuli within 0.2 s (Andreassi, 1995). Recently, we (Reinhard and Lachnit, 2002) utilized the phenomenon that frequently presented stimuli lead to smaller pupillary dilations (Friedman et al., 1973) as a tool in the examination of compound processing and rule learning in RT tasks. Hence, pupillary dilation may be used in order to investigate compound processing and the utilization of rules in discrimination problems in RT tasks as well as in Pavlovian conditioning procedures. Thus, the differential conditioning of anticipatory pupillary dilation demonstrated in the present four experiments enhances our arsenal of methods for the examination of processing of compound stimuli.

Acknowledgements This research is part of the doctoral thesis of Gu¨ nter Reinhard and was supported by grant DFG LA 564/8-1 from the National German Science Foundation (Deutsche Forschungsgemeinschaft) to Harald Lachnit. We thank John Furedy and an anonymous reviewer as well as Ottmar Lipp for helpful comments on earlier drafts of this manuscript. We thank also Torsten Hoffmann and Tilo Ko¨ nig for assistance with data collection.

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