52(2014), (2015),••–••. 509–517. Periodicals, Inc. Printed the USA. Psychophysiology, •• WileyWiley Periodicals, Inc. Printed in the in USA. C 2014 Society for Psychophysiological V Psychophysiological Research Research Copyright © 10.1111/psyp.12372 DOI: 10.1111/psyp.12372

Modulation of eyeblink and postauricular reflexes during the anticipation and viewing of food images

KAREN R. HEBERT,a,b FERNANDO VALLE-INCLÁN,c and STEVEN A. HACKLEYa a

Department of Psychological Sciences, University of Missouri–Columbia, Columbia, Missouri, USA Department of Occupational Therapy, University of Missouri–Columbia, Columbia, Missouri, USA c Department of Psychology, University of La Coruña, La Coruña, Spain b

Abstract One of the goals of neuroscience research on the reward system is to fractionate its functions into meaningful subcomponents. To this end, the present study examined emotional modulation of the eyeblink and postauricular components of startle in 60 young adults during anticipation and viewing of food images. Appetitive and disgusting photos served as rewards and punishments in a guessing game. Reflexes evoked during anticipation were not influenced by valence, consistent with the prevailing view that startle modulation indexes hedonic impact (liking) rather than incentive salience (wanting). During the slide-viewing period, postauricular reflexes were larger for correct than incorrect feedback, whereas the reverse was true for blink reflexes. Probes were delivered in brief trains, but only the first response exhibited this pattern. The specificity of affective startle modification makes it a valuable tool for studying the reward system. Descriptors: Food cues, Reward, Startle blink, Postauricular reflex, Anticipatory attention

but were allowed to eat a piece of chocolate following a correct response. Probe white noise bursts were delivered just before the participant reached into a briefly illuminated box for the reward or punishment. Extensive prior research has shown that the eyeblink (orbicularis oculi) component of startle is enhanced during unpleasant emotions (Lang, Bradley, & Cuthbert, 1990, 1997), whereas the postauricular (pinna-flexion) component is larger during pleasant ones (Benning, Patrick, & Lang, 2004). Congruent with those findings, we observed potentiation of the blink reflex during anticipation of banana peel consumption, but enhancement of the postauricular reflex (PAR) response during anticipation of the chocolate. This finding is intriguing in that it appears to contradict studies in which the blink reflex was elicited during anticipation of positive versus negative photographs. Across several studies, a quadratic pattern has consistently been observed, in which blink reflexes evoked during anticipation of pleasant and unpleasant images are more or less equally enhanced as compared to when neutral slides are expected (e.g., Dichter, Tomarken, & Baucom, 2002; Lipp, Cox, & Siddle, 2001; Mallan, Lipp, & Libera, 2008; Sabatinelli, Bradley, & Lang, 2001). An obvious explanation for the discrepancy would be that real rewards and punishments activate motivation-related pathways more robustly than do photographs. However, the study by Sabatinelli and colleagues involved presenting images of attractive nude women and menacing snakes to young men with snake phobia. The anticipatory emotions felt by Sabatinelli’s participants might very well have been more intense than those experienced by our subjects. A second potential explanation of the discrepancy is that significant anticipatory effects might be obtained only when the

According to the Center for Disease Control, approximately one third of all Americans are currently obese and another third are overweight (Flegal, Carroll, Ogden, & Curtin, 2010). In response to this public health concern, neuroscientists are exploring the role of the reward system in overconsumption of food. The heart of the reward system is the mesencephalic dopamine pathway, the activity of which is theorized to encode an error signal in reward prediction (Schultz, 1998, 2002). More specifically, dopaminergic cell discharge is assumed to reflect the difference between favorability of the actual outcome of a motor response as compared to the outcome that was anticipated. The focus of recent efforts at this laboratory has been on developing psychophysiological measures of this anticipation (reviewed in Hackley, Valle-Inclán, Masaki, & Hebert, 2014). A study of immediate relevance examined the question of whether anticipation of food rewards and punishments is characterized by hedonic valence (Hackley, Muñoz, Hebert, Valle-Inclán, & Vila, 2009). The study was motivated by findings of anticipatory valence effects reported in fMRI (functional magnetic resonance imaging) research on tasks with monetary rewards and punishments (reviewed in Knutson & Greer, 2008). Our participants performed a perceptual categorization on each trial. They were required to chew on a banana peel segment after making an error,

A preliminary report of these findings was presented at the 2012 meeting of the Society for Psychophysiological Research and comprised a portion of the first author’s doctoral dissertation (2012). Address correspondence to: Karen R. Hebert, Department of Occupational Therapy, 805 Clark Hall, University of Missouri–Columbia, Columbia, MO 65211, USA. E-mail: [email protected]

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510 2 positive and negative outcomes are response contingent. In the fMRI studies reviewed by Knutson and Greer (2008) and in the blink/PAR study of Hackley and colleagues (2009), the pleasant and unpleasant stimuli comprised rewards and punishments for correct and incorrect responses, respectively. By contrast, in the picture-anticipation studies that failed to produce a valence effect on the blink reflex (e.g., Dichter et al., 2002; Sabatinelli et al., 2001), the pleasant and unpleasant stimuli were given gratuitously. We know from fMRI (Tricomi, Delgado, & Fiez, 2004) and event-related potential (ERP; Masaki, Yamazaki, & Hackley, 2010) studies of reward anticipation that response contingency does matter. Using a task similar to the one employed in the present study, Masaki and colleagues found that a cortical ERP specific to reward/punishment anticipation (the stimulus-preceding negativity, SPN) was twice as large when participants were led to believe that monetary gains and losses were dependent on their guessing responses than when they correctly understood that these outcomes were purely random. A third possible explanation of the discrepancy is that valence effects in the Hackley et al. (2009) experiment might not have reflected anticipation of food consumption but, rather, receipt of performance feedback. Startle probes were delivered 5–7 s after onset of task feedback, 1–2 s prior to display of the go signal for eating. This signal to consume the food conveyed no new information because the participants already knew whether they had responded correctly or incorrectly. Thus, the positive or negative emotion inferred from startle modulation might have been a hedonic reaction to the performance feedback, rather than an anticipatory state. These issues were examined in the present study using a blocked design in which either rewards were given for correct responses or else punishments were delivered for incorrect responses, but not both. The rewards were photographs of appetizing, high-fat or sugary food and the punishments, images of contaminated or culturally objectionable food. During negative blocks, the participant received a neutral picture (dull, but edible food) if their guess was right, but a photo of disgusting food if their guess was wrong. During positive blocks, a neutral picture meant that the guess was incorrect, whereas a photo of delicious food indicated a correct key-press response. Similar chance-to-win versus chanceto-lose manipulations have documented valence-specific anticipation effects in fMRI research (Knutson & Greer, 2008). In the present study, white noise probes were delivered on some trials after the participant entered a response but before feedback was delivered (i.e., anticipation); on other trials, probes were delivered while the feedback slide was viewed (receipt). Suppose that, during the anticipatory interval, postauricular reflexes are enhanced for positive relative to negative trial blocks but the reverse is true for blink reflexes. Such a pattern could suggest that anticipation of pleasant or unpleasant stimuli does have a hedonic tone, at least if these stimuli are contingent on one’s voluntary action, that is to say, if the stimuli are perceived as being rewards or punishments. Analysis of reflex modulation during the receipt interval allows independent assessment of top-down and bottom-up aspects of reinforcement. Suppose that modulatory effects are driven solely by information regarding whether the response was correct or incorrect, as defined for the specific task. If so, we would expect potentiation of the PAR during the feedback slides on correct trials and potentiation of the blink reflex on incorrect trials, regardless of whether the trial block was negative (included disgusting images) or positive (included appetizing images). But suppose, on the other hand, that reflex modulation is driven solely by the intrinsic

hedonic valence of the slides. If so, there should be no effect of correctness, per se. Rather, there should be an interaction of block type and feedback correctness, such that startle modulation only occurs when disgusting or delicious food photographs are viewed. Our experiment also addressed a methodological issue. Because habituation and refractory effects on the PAR are very modest, it should be possible to present probes more frequently than in the typical affective-modulation study. The relative refractory period is 90–100 ms for the PAR (Fox, Peyton, & Ragi, 1989; Yoshie & Okudaira, 1969). For the acoustic blink reflex, the relative refractory period is at least an order of magnitude greater, as assessed either in terms of response amplitude (Fox, 1978) or response probability (Dodge, 1913, in a pioneering study that employed optical recordings of eyelash movement with ms-level precision). Habituation of the blink reflex is rapid and long lasting (Ornitz & Guthrie, 1989), which imposes a practical limit on the number of trials per session to somewhere between 60 and 80. By contrast, habituation of the PAR is so slight that responses to several thousand probes can be measured in a single session (Hackley, Woldorff, & Hillyard, 1987; see Caeser, Ostwald, & Pilz, 1989, for habituation data in rats). The ability to present more probes per trial and more trials per subject should enhance the efficiency of startle research. Ultimately, it might be possible to develop paradigms with adequate power for clinical tests of affective disorders. To examine this issue, we presented startle stimuli in brief trains of six probes with an onset asynchrony of 115 ms, near the edge of the PAR refractory period. A measurable blink reflex would be expected only for the first probe, but a PAR should be observed following each of the six white noise bursts. Method Participants In the main experiment, analyses of the PAR were based on 51 young adult undergraduates (31 female), and analyses of the blink reflex, on 55 undergraduates, of whom 34 were female. All were recruited from an introductory psychology class. Individuals who were actively attempting to lose weight or who reported an eating disorder were excluded, because of concerns that they might react defensively to photos of delicious food (“frustrative nonreward,” Drobes et al., 2001). We asked that participants refrain from eating for 1 hr prior to arriving at the laboratory, so that they would not be fully sated while viewing the images. The 51 or 55 subjects who provided an adequate number of artifact-free reflexes were chosen from an original sample of 60. Twelve additional participants (6 female) were recruited for a pilot study to select optimal slide stimuli. Ethical approval by the Campus Institutional Review Board and written informed consent from each participant was obtained for this research. Photographs of Food For the pilot study, 150 slides showing appetitive, neutral, and disgusting food items downloaded from the Internet were evaluated. There were 50 slides in each category. Appetitive images included sugary or high-fat foods such as ice cream, chocolate cake, cookies, cupcakes, sliced fruit, candied apples, tacos, cheeseburgers, fried chicken, and pizza. Neutral photographs showed healthy but bland foods such as uncooked vegetables (e.g., cauliflower, broccoli, zucchini, kale), prepared but very plain-looking starches (whole

PAR PAR and food rewards wheat bread, pita, grits, ramen noodles, rice, sliced bagels), and uncooked beans (mung, northern white). The disgust category comprised food items that were either contaminated or else culturally objectionable within the population from which our sample of participants was drawn. Slides in the disgust category portrayed such items as roasted dogs and rats, moldy bread, rotten apples and peaches, fish heads with prominent teeth and eyes, baked grub worms, maggot-infested pork, stewed organs, and moldy hot dogs. After viewing each slide for 3 s, participants rated on a 1–9 scale: (a) How appetizing did the food look? and (b) How much did the item look like food? The latter question was necessary because some pictures in the disgust category were difficult to immediately identify. Presentation order for the 150 slides was random. Higher scores indicated greater palatability and ease of recognition. Our focus in this study was strictly on palatability of the food; participants were not asked to introspect on how the photographs made them feel (e.g., dominant, excited, happy). Selection of the stimulus sample for the final study was based on two criteria: First, images were only included if the averaged “looks like food” score was greater than three, a cutoff near the mean for the original set of 50 disgusting slides (M = 3.2). Second, distinct affective categories were created using palatability ratings (appetitive: 6–9, neutral: 3–6, and disgusting: 1–3). Of the images selected for the main study, average palatability was 6.5 for the appetitive, 4.2 for the neutral, and 1.7 for the disgusting slides. Participants in the main experiment evaluated the slides immediately after the electrophysiology session using a different, more standard set of scales. The 32 appetitive, 48 neutral, and 16 disgusting images that had previously been viewed were rated with respect to valence (unhappy to happy) and arousal (calm to exciting). This was done using a computerized version of the Self-Assessment Manikin (SAM; Bradley & Lang, 1994; Lang, Bradley, & Cuthbert, 2005). Cartoon figures in that scale represent numeric values ranging from 1–9. Design, Procedure, and Apparatus On each trial of the guessing game, participants saw a pair of empty boxes, one to the right and one to the left of fixation (Figure 1). The boxes were filled 500 ms after onset with either a red circle or a red square. In trial blocks for which there were circles inside the boxes (positive blocks), participants were to guess where the “delicious” food was hiding. When there were squares in the boxes (negative blocks), they were to guess where the “dull” food was hiding and avoid the disgusting food. The z and m keys were used to select the

5113 left and right boxes, respectively. As soon as the response was registered, the fixation cross was replaced with an arrow for 200 ms, indicating the selected box. This was followed by a blank screen lasting 6 s (the anticipation period), and then by a 6-s long display of the feedback picture (the receipt period). After picture offset, a brief RSVP (rapid serial visual presentation) task was administered. This unrelated procedure was intended to minimize affective carryover and help maintain the participant’s alertness. Nine randomly selected letters were presented at fixation. Fifty percent of the time, a digit (1–9) was substituted for a letter within the third through the seventh locations. Participants responded at the conclusion of the sequence by entering that digit on the keyboard or a zero if no number was observed. Trials lasted approximately 15 s, as measured from onset of the empty boxes until conclusion of the RSVP task. Intertrial intervals ranged from 6 to 8 s. Startle probes were randomly presented during anticipation (4.5 or 5 s after offset of the arrow) on one half of the trials and during feedback receipt (4 or 5 s after slide onset) on the other half. Two no-probe trials were included in each block of 24 to reduce predictability of the startle stimuli. Prior to the guessing task, five white noise bursts were presented to familiarize participants with these stimuli and partially habituate the startle response. There were four blocks of 24 trials. Positive and negative blocks were counterbalanced across participants using an A-B-B-A design. On positive blocks, there were always 16 appetitive slides, signaling a correct key-press response, and eight neutral slides, indicating an incorrect response. On negative blocks, there were 16 neutral slides (correct) and eight disgusting slides (incorrect). Correct feedback was presented more frequently than incorrect to encourage participants to believe that the feedback was response contingent and that they were successfully learning the task (Masaki et al., 2010). As a manipulation check, participants were asked on a postexperimental questionnaire to rate with respect to a 1- to 7-point scale: (a) “How competent did you feel at being able to guess the food you were going to see?” and (b) “To what degree did there seem to be a pattern to the correct responses?” In the slide-rating task, the 96 food pictures that had been seen in the electrophysiology session were presented again in random order. Each picture was displayed for 3 s, followed by the standard 1–9 scales for valence and arousal. Participants were instructed to view the image for the entire 3 s and then make their ratings based on how they felt while looking at the image. All images were presented on a 32 × 23 cm computer monitor positioned approximately 50 cm in front of the participant. The

Figure 1. Schematic portrayal of a sample trial from a positive “chance-to-win” block with correct feedback (i.e., delicious food).

512 4 displays were controlled using MATLAB software in conjunction with Psychophysics Toolbox (Brainard, 1997; Pelli, 1997). Physiological responses were recorded on a separate computer running NeuroScan acquisition software (NeuroScan, Inc., Herndon, VA). Arousal and valence ratings were obtained with E-prime 2.0 software (Psychology Software Tools, Pittsburgh, PA). Reflex Elicitation and Recording During the electrophysiology session, startle probes were presented binaurally through insert earphones at an intensity of approximately 90 dB (SPL-A) and a rise/fall time that was nearly instantaneous. These 40-ms long, white noise bursts were delivered in trains of six, with an onset asynchrony of 115 ms (i.e., a 40–75 duty cycle). As noted above, studies of habituation and refraction led us to expect that only the first probe would evoke a blink reflex, whereas all six would trigger PARs. Surface electromyograms (EMG) were recorded from the left and right orbicularis oculi and retrahens auriculam muscles (Blumenthal et al., 2005). To locate the latter, the pinna was gently pulled forward until the muscle and tendon were visible as a wedge-shaped protrusion overlying the mastoid process (Sollers & Hackley, 1997). One 0.35-cm Ag-AgCl electrode was placed over the belly of the muscle. Deviating from standard practice (Blumenthal et al., 2005), we placed the second electrode on the rear wall of the pinna. There is a polarity inversion of the PAR at this site (O’Beirne & Patuzzi, 1999); therefore, positioning the inverting electrode here should double the amplitude of the observed voltages. We confirmed that this was the case in a test subject, but the extent to which the signal-to-noise ratio is actually improved by this derivation remains to be determined. Vertical and horizontal electrooculograms (EOG) were also recorded in our study using standard periorbital sites. Prior to electrode placement, the skin was rubbed with a mild abrasive gel to obtain impedances below 10 kΩ. Raw EMG signals for each ear and eye were continuously recorded at a sampling rate of 600 Hz. The EMG data were filtered online with a band-pass of 3–300 Hz. The EOG recordings had a band-pass of .01–30 Hz. The EMG data were rectified offline using EEGLAB software (Delorme & Makeig, 2004). These data streams were then segmented to create 1,000-ms analysis epochs that began 100 ms prior to onset of the first probe of each train. Analysis of reflex EMG at our laboratory is based on multichannel, ERP-style, signal averaging. While this method has advantages, we wish to point out that the conventional, wellestablished methods of startle blink research can also be employed to study affective modulation of the PAR (e.g., Sandt, Sloan, & Johnson, 2009). The investigator simply needs to keep in mind that responses of the postauricular muscle are smaller and more rapid than those of the orbicularis oculi muscle, and that the participant must maintain a posture with some degree of tension in the neck region. Data Processing The signal-to-noise ratio of the PAR is only slightly more favorable than that of the largest event-related brain potentials. In order to identify an optimal set of trials for analysis, we adapted a method that was originally designed to measure P300 on single trials (Kutas, McCarthy, & Donchin, 1977). In the first step, epochs obscured by movement artifact or amplifier blocking were eliminated. Next, the left and right EMG channels were averaged

K.R. an, and K.R.Hebert, Hebert, F. F. Valle-Incl Valle-Inclán, and S.A. S.A. Hackley together. A preliminary, pattern-recognition template was then generated by signal averaging the entire data set (N = 60 participants), collapsing across conditions. This template was then correlated with the observed EMG on each trial, which was first smoothed using a 9-point boxcar filter. If the correlation coefficient was greater than 0.20, the trial was accepted. (Preliminary tests showed that this cutoff provided a good balance of quantity vs. quality of data.) Subjects were accepted if there were at least 30 total trials, with a minimum of two in each condition. Data from the selected trials and participants were then reaveraged, creating a new template. The single-trial correlation procedure was repeated for this subset of the data, yielding the final sample of subjects and trials. Ultimately, the average number of accepted trials per condition for PAR was 18.6 (39% of 48) during positive and negative feedback-anticipation conditions, 10.4 (65% of 16) during positive and negative correct feedback-receipt epochs, and 5.3 (66% of 8) during positive and negative incorrect feedback-receipt epochs. For blink reflex, the corresponding values were 15.4 (32%), 12.5 (78%), and 6.4 (80%). Head movements, spontaneous blinking, and large gaze deviations were more common during anticipation than receipt of feedback. The selected trials were then reaveraged, separately for each condition and participant, using rectified but unsmoothed EMG epochs. Postauricular responses to the six probes were quantified separately for each condition. Amplitudes were measured at the point in time (±1.6 ms) corresponding to peak voltage in the grand average waveform and then entered into the repeated measures analysis of variance (ANOVA). The blink EMG data were handled similarly, except that only the response to the first probe was measured. No modulation of onset latency for the PAR or blink reflex was evident in the grand average waveforms, so a formal analysis of these measures was not undertaken. Statistical Analyses The amplitudes of reflexes elicited during anticipation and receipt periods were analyzed separately. To maximize comparability with previous findings, our primary analyses, which were hypothesis driven, focused on the response to the first probe within each train. Secondary analyses, which were exploratory, included the factor probe number (or peak, for short) when assessing PAR amplitude. Main effects and interactions involving this 6-level variable were corrected for violations of the sphericity assumption using the Huynh and Feldt (1970) method. Preliminary analyses that used recording side (left vs. right eye or ear) as a factor yielded no main effects or interactions. Therefore, we collapsed across this variable in the data reduction procedure, as described above. GreenhouseGeisser corrected F values are reported for all significant omnibus tests. A rejection region of α < .05 was adopted for all statistical tests. The ANOVA that assessed amplitude modulation during anticipation had a single factor block type, with two levels, positive (chance to win) and negative (chance to lose). On the assumption that the null results obtained in previous research (e.g., Dichter et al., 2002; Lipp et al., 2001; Mallan et al., 2008; Sabatinelli et al., 2001) were due to the failure to make anticipated pleasant and unpleasant outcomes contingent upon the participant’s voluntary motor response, we predicted differences in reflex amplitude across conditions. Our previous results using a paradigm with responsecontingent rewards and punishments (Hackley et al., 2009) led us to predict that the PAR would be larger in positive than negative blocks, whereas the reverse would be true for the blink reflex.

PAR PAR and food rewards

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For analyses of the receipt period, within-subject factors of the 2 × 2 ANOVA were block type (positive and negative) and feedback correctness (correct vs. incorrect). Because committing an error is unpleasant and sometimes has dangerous consequences, negative feedback potentiates startle blink (Riesel, Weinberg, Moran, & Hajcak, 2013; Skolnick & Davidson, 2002). Therefore, we expected larger blink reflexes during incorrect than correct feedback displays. Noting that one’s action has yielded a favorable outcome is usually a pleasant experience; therefore, we predicted the reverse pattern of modulation for the PAR. This relationship has not been examined before, to our knowledge. The photographs used in our feedback displays had intrinsic hedonic quality, independent of their instructionally defined meaning. Consequently, we expected to observe, superimposed upon any modulatory effect of correctness, variations in reflex amplitude due to the well-documented effects of viewing emotioninducing images. Specifically, we predicted that the blink reflex would be suppressed as delicious food was viewed (e.g., Bradley, Codispoti, Cuthbert, & Lang, 2001), but that the PAR would be potentiated (Sandt et al., 2009). Disgusting food images have not previously been studied, but we expected the reverse pattern for these unpleasant stimuli. This pattern of affective modulation would be detected in our 2 × 2 design as an interaction of correctness and block type, with post hoc analyses needed to confirm the direction of effects.

Figure 2. Picture ratings by arousal and hedonic valence.

Postauricular Reflex Modulation Results Subjective Ratings Participants indicated that they believed to a moderate degree in the predictability of trial outcomes (M = 3.97, on the 1–7 scale, SD = 1.7) and in their ability to guess which food picture would be displayed (M = 4.15, SD = 1.5). As in the previous similar study by Masaki and colleagues (2010), our instructions convinced participants that the feedback was to some extent contingent on their response. Valence and arousal ratings were analyzed to confirm that the slides induced the intended emotional response. Appetitive pictures (M = 7.39, SD = 0.23) were rated as more pleasant than neutral (M = 4.23, SD = 0.26) or disgusting photos (M = 1.51, SD = 0.16), and neutral images were more pleasant than disgusting images, F(2,112) = 640.23, p < .001 (see Figure 2). Additionally, appetitive pictures (M = 6.40, SD = 0.30) were rated as more arousing than neutral (M = 3.07, SD = 0.26) or disgusting ones (M = 3.85, SD = 0.40). Contrary to the general rule that emotion-inducing pictures are arousing (Cuthbert, Bradley, & Lang, 1996), the disgusting images were not rated higher in this regard than neutral images, at least not significantly so. To our knowledge, disgusting food images have not previously been examined in startle research. However, spoiled food pictures have been included in the category of “contamination,” which yields very modest arousal ratings and skin conductance values that are at control levels (Bradley et al., 2001). Low arousal values for the repulsive food images can be expected to weaken bottom-up modulation of the blink reflex (Bradley et al., 2001), but to have no impact on the PAR. The two previous experiments that examined this issue reported that the arousal value of unpleasant slides or videos had no influence on PAR amplitude (Gable & Harmon-Jones, 2009; Sparks & Lang, 2010). Affective modulation of the PAR is quite specific to positive emotions.

Trains of white noise bursts proved to be effective reflex elicitors. Inspection of the raw data indicated that, if one PAR was evident on a trial, then five or six EMG bursts in rapid succession were usually visible. Morphology and peak latency conformed to typical patterns for this reflex (Hackley, 1993), both in the unrectified, singletrial data and in the rectified, signal-averaged waveforms. To maximize comparability with previous research, especially our previous study of reward anticipation (Hackley et al., 2009), the primary analysis included only the response to the first white noise burst of each train. During the anticipation period, no effect of block type on amplitude or baseline was observed, F(1,50) < 1, n.s.; t(50) = −.87, p = .38, respectively. The nearly perfect superposition of the two waveforms is evident in Figure 3. This null effect is contrary to our previous findings, in which probes were delivered after feedback but prior to reward or punishment. In the analysis that included probe number as a factor, there was also no reliable difference between positive and negative blocks. However, variation in amplitude across the six responses was significant, F(5,250) = 17.9, p < .0001, mainly due to reduction in the size of the second peak (Figure 3). This nonmonotonic decline was also observed during the feedback receipt period, F(5,250) = 8.4, p < .0001. Analysis of the PAR to the first probe during the feedback receipt period included within-subject factors of block type and feedback correctness. A main effect for correctness was noted, such that larger EMG bursts were evoked as the participant learned that their key-press response had been correct, F(1,50) = 12.1, p < .002 (see Figure 3). When six probes were included in the analysis, an interaction of feedback correctness and probe number was obtained, F(5,250) = 5.7, p < .0001 (Figure 4). Inspection of the means suggests that enhancement of the PAR during correct feedback was largest for the first peak, but absent or slightly reversed for the others. There was no three-way interaction of block type, feedback correctness, and probe number, F(5,250) < 1, in our study. Neither

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Figure 3. Grand-average orbicularis oculi and postauricular EMG responses during the anticipation period for positive “chance-to-win” and negative “chance-to-lose” blocks.

was there a two-way interaction of block and correctness either in the analysis of six peaks, F(5,250) = 2.8, p = .59, or that of the first peak, F(1,50) = 1.9, p = .16. This suggests that the intrinsic motivational quality of the pictures (delicious vs. disgusting) contributed little to PAR modification in the present study. Suppression of affective startle modulation in the context of cognitive task performance has previously been noted (Panayiotou, van Oyen Witvliet, Robinson, & Vrana, 2011). Blink Reflex Modulation As expected, only the first probe within the train reliably elicited a blink reflex. Inspection of the single-trial data indicated that occasionally the first white noise burst failed to elicit a blink, but the second or third one did. Our analyses were restricted to the reflex elicited by the first probe. Consistent with findings of previous slide-expectation studies reviewed earlier, there was no enhancement of reflex amplitudes on negative as compared to positive blocks during the anticipation interval, F(1,54) = 1.18, p = .28. In fact, the nonsignificant trend (Figure 3) was in the opposite direction (see also Sabatinelli et al., 2001, Footnote 2). There was also no significant difference in baseline EMG levels, t(54) = .25, p = .80.

Figure 4. Modulation of the blink (left graph) and postauricular (right graph) responses during receipt of correct versus incorrect task feedback.

During the receipt period, blinks that were elicited as unfavorable feedback was processed were larger than those elicited during favorable feedback, F(1,54) = 5.9, p < .02 (Figure 4). There was no interaction of block type with correctness, F(1,44) < 1, congruent with the PAR results. This suggests that intrinsic emotional salience of the slides had little impact in this cognitively oriented task (see also Panayiotou et al., 2011). Discussion We found no significant effect of negative versus positive block type on the blink reflex or PAR during anticipation of picture viewing. To maximize statistical power, we included only these two anticipatory conditions. A neutral control condition, with both delicious and disgusting images as feedback, would have allowed arousal and attention effects to be assessed. In such a condition, attention would presumably have been more visually focused because all anticipated displays would have been emotionally salient. Attention and arousal have complex effects on the PAR and blink reflex (e.g., Hackley, 1993; Lipp, Cox, & Siddle, 2001), effects that certainly merit further investigation. However, the focus of the current study was on detecting the presence of valencespecific modulation during anticipation and receipt of feedback. The null effect of valence during feedback anticipation for the PAR is new, but results for the blink reflex are in agreement with previous studies involving the expectation of emotionally evocative images (e.g., Dichter et al., 2002; Lipp et al., 2001; Mallan et al., 2008; Sabatinelli et al., 2001). How can these null effects be reconciled with our previous finding of PAR and blink reflex modulation during anticipation of food (Hackley et al., 2009) and with the extensive fMRI literature concerning anticipation of monetary rewards and penalties (Knutson & Greer, 2008)? The most obvious explanation is based on the relative hedonic value of real versus simulated rewards. Previous studies demonstrating that anticipation is associated with an objectively measurable affective valence have used tangible objects to manipulate affect (namely, money, food, or electric shocks). Further support for this interpretation comes from the weak or inconsistent valence effects obtained during imagery. When startle probes are presented while participants imagine or remember emotional scenes, potentiation of the blink reflex is sometimes observed not just for unpleasant scenarios but also for pleasant ones (Cook, Hawk,

PAR PAR and food rewards Davis, & Stevenson, 1991; Miller, Patrick, & Levenston, 2002; Vrana & Lang, 1990; Witvliet & Vrana, 1995). This purely quadratic pattern, as it is known, indicates that the modulatory effect is not valence specific. There is a good argument against the real versus simulated explanation. As noted earlier, it seems likely that the anticipatory affect experienced by participants in the Sabatinelli et al. (2001) study would have been stronger than that of our study (Hackley et al., 2009). We suspect that the young men in the Sabatinelli experiment might have preferred looking at photos of beautiful women to eating tiny pieces of chocolate and, given their phobia, would have found viewing pictures of menacing snakes to be quite aversive. Direct comparison of the anticipation of real, imminent rewards/punishments with that of photographs will be needed to resolve this issue. The low intensity of some of the emotion-inducing images used in the present stimulus may have undermined the effectiveness of our bottom-up manipulation of valence, at least with regard to the blink reflex. Modulation of this component of startle is greater for more arousing stimuli, whether pleasant or unpleasant (Cuthbert et al., 1996), and photographs of food are typically not the strongest blink-reflex modulators (Bradley et al., 2001). Fortunately, this issue is of less concern with respect to the PAR. Appetizing food pictures do seem to be, in fact, the most effective stimuli for modulating this component of startle (Sandt et al., 2009). Also, as noted earlier, the arousal value of negative emotion-inducing stimuli apparently has no influence on PAR amplitude (Gable & Harmon-Jones, 2009; Sparks & Lang, 2010). Furthermore, anticipatory affect during the period in which feedback was awaited should have been influenced not just by the bottom-up, intrinsic quality of the slides. It should also have been determined by the top-down, instructionally defined condition: chance-to-win versus chance-to-lose in the guessing game (Knutson & Greer, 2008). The present data support a revised interpretation of the rewardanticipation data of Hackley and colleagues (2009). Probes were presented in that study as subjects awaited the go signal for eating, but after they had received feedback regarding correctness of their key press. It is well established (Riesel et al., 2013; Skolnick & Davidson, 2002) that receipt of positive or negative responsecontingent feedback modulates the blink reflex in a manner similar to viewing pleasant or unpleasant photographs. The present data show that this is also true for the PAR. Thus, it now seems likely that our 2009 findings did not reflect anticipatory affect but, rather, an emotional response to the valence of the performance feedback. Relation of PAR to Startle Blink There is now an empirically supported model detailing the relation of the pinna-flexion reflex arc to the main neuroanatomical pathway for startle (Horta-Junior, Lopez, Alvarez-Morujo, & Bittencourt, 2008). The Lopez group has determined that the most direct stimulus-response path is disynaptic, extending from the cochlear root nucleus to the medial facial motor nucleus. However, a side path does include the startle center, the nucleus reticularis pontis caudalis. Differential sensitivity to refraction and prepulse inhibition within the two paths might have contributed to the Correctness × Probe Number interaction observed in the present data set. There is also a theory that attempts to explain why affective valence modulates the human PAR in a direction opposite that of eyeblink, leg flexion, and other components of startle. That theory (Johnson, Valle-Inclán, Geary, & Hackley, 2012) takes as its start-

5157 ing point the fact that the PAR is wholly vestigial in our species. It is vestigial in the sense that it is too weak to generate physical movement of the pinnae (even in people who can voluntarily move their ears) and, therefore, it can serve no adaptive function. An explanation for the paradoxical effects of emotion must lie within phylogeny. Johnson and colleagues point out that infant mammals with large ears pull them back during nursing in order to comfortably position the head. Pinna retraction is assumed to signal the infant’s intention to suckle, triggering the mother’s let-down reflex. Natural selection is therefore postulated to have created circuits to prime the ear-retraction motor pathway when appetitive cues (i.e., food or exposed breasts) are observed. The circuit survives in our species, priming the ear-retraction musculature during appetitive states. If an abrupt and intense noise happens to be registered during this primed state, the PAR will be enhanced to an extent that overrides the normal pattern of affective startle modulation. Wanting Versus Liking Such anatomical and functional theories are vital if the PAR is to be integrated within the literature of affective neuroscience and ultimately find practical application in the clinic. The present data speak to a specific issue within affective neuroscience, the partition of reward processes into two separate components, wanting and liking (Berridge & Robinson, 1998, 2003). The wanting or incentive salience component is manifested during stimulus approach and goal-directed action to obtain a reward. It is mediated by the mesostriatal dopaminergic system and is strongly related to the incentive value of reward. Extensive research (Berridge & Robinson, 1998, 2003) has shown that when the dopamine system is disrupted by neuropharmacological manipulations, humans and other species show decreased effort to obtain a reward but still display normal liking of it upon receipt. Circuits that involve opioid neurotransmitters, particularly within the nucleus accumbens, are important in mediating this hedonic impact. The principal objective measure of liking is valence-specific facial expressions. Because social factors can diminish or bias overt facial responses in adult humans, their value is limited. Furthermore, in many situations requiring approach behavior, incentive salience and hedonic impact can overlap in time making the two processes difficult to distinguish. For these reasons, it would be useful to determine if affective startle modulation constitutes a pure index of either wanting or liking. The weight of available evidence suggests that valence effects on the blink reflex component of startle specifically reflect hedonic impact liking. As discussed earlier, mere anticipation of a pleasant versus an unpleasant stimulus, while important for incentive approach, does not reliably modulate the blink reflex (e.g., Sabatinelli et al., 2001). Our previous anticipation study (Hackley et al., 2009) raised concerns that this conclusion might not be valid for all components of startle. The present findings allay those concerns. Valence effects on both blink reflex and PAR were observed during receipt of task performance feedback but not during anticipation. Taken together, these findings suggest that affective startle modulation could be a useful means of disentangling wanting versus liking processes that occur during approach behaviors in humans. Rapid Data Acquisition We conclude with a methodological point. Affective startle experiments are typically slower paced than ERP or fMRI studies, and

K.R. an, and K.R.Hebert, Hebert, F. F. Valle-Incl Valle-Inclán, and S.A. S.A. Hackley

516 8 they require a much larger sample size. This is at least partly because the eyeblink component of startle has a long refractory period and habituates to low levels in only 80 or so trials. By contrast, the PAR has a resistance to habituation and a refractory period that is comparable to that of the fastest brain stem ERPs (Yoshie & Okudaira, 1969). Using brief trains of startle pulses, we showed that several postauricular reflexes could be recorded during a single trial. However, affective modulation was only reliable for the first, least refractory response. Borrowing from the steady-state ERP literature, Aaron and Benning (2014) presented 65 dB clicks every 100 ms throughout affective image viewing. They observed robust PAR modulation in

the expected direction during the middle and later portion of the slide-viewing interval. In our study, the absence or slight reversal of modulation for Probes 2–6 might have been due to refraction, prepulse inhibition, or middle ear muscle activation. We are examining this topic in ongoing research. Aaron and Benning’s success with high-density probes suggests that an effective methodology might be to turn on a repetitive click at the beginning of an experimental session, and just leave it on. The PAR recording would offer a continuous, albeit punctate, psychophysiological measure similar to the steady-state visual ERP. With more data per session and fewer participants per experiment, startle research could make rapid progress.

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