PSYCHOLOGICAL SCIENCE. Special Section

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1 Special Section CLASSICAL DELAY EYEBLINK CONDITIONING IN 4- AND 5-MONTH-OLD HUMAN INFANTS Dragana Ivkovich, 1,2 Kimberly L. Collins, 1 Carol O. Eckerman, 1 Norman A. Krasnegor, 3 and Mark E. Stanton 2 1 Duke University, 2 U.S. Environmental Protection Agency, and 3 National Institute of Child Health and Human Development Abstract Simple delay classical eyeblink conditioning, using a tone conditioned stimulus (CS) and airpuff unconditioned stimulus (US), was studied in cross-sectional samples of 4- and 5-month-old healthy, full-term infants. Infants received two identical training sessions, 1 week apart. At both ages, infants experiencing paired tones and airpuffs demonstrated successful conditioning over two sessions, relative to control subjects who had unpaired training. Conditioning was not evident, however, during the first session. Two additional groups of 5- month-olds received varied experiences during Session 1, either unpaired presentations of the CS and US or no stimulus exposure, followed by paired conditioning during Session 2. Results from these groups suggest that the higher level of conditioning observed following two sessions of paired conditioning was not the result of familiarity with the testing environment or the stimuli involved but, rather, the result of retention of associative learning not expressed during the first conditioning session. Address correspondence to Dragana Ivkovich, Department of Psychology: Experimental, Duke University, Durham, NC ; dragana@acpub.duke.edu. Classical eyeblink conditioning has become a successful paradigm for studying cognitive and neural processes underlying learning and memory in several species, including humans. The majority of human studies have involved young or aged adult populations (e.g., Kimble & Pennypacker, 1963; Solomon & Pendlebury, 1988; Spence, 1953; Woodruff-Pak & Thompson, 1988). There have been relatively few studies of human infants and young children despite their potential for yielding fundamentally important information about the early development of cognitive and neural processes underlying associative learning, and despite the potential for eyeblink conditioning to become an important tool for assessing neurocognitive development in infants at high risk for neurobehavioral disorders. A few researchers have reported successful eyeblink conditioning in human infants and children (e.g., Fitzgerald & Brackbill, 1976; Little, Lipsitt, & Rovee-Collier, 1984; Ohlrich & Ross, 1968), but little parametric work has been conducted, and none of the work traces the development of eyeblink conditioning phenomena over a substantial span of human infancy. Recently, we have been developing procedures for studying eyeblink conditioning in 4- and 5-month-old human infants. Eventually, we expect that these procedures can be adapted for use over a broader age range, be used with infants at high risk for central nervous system damage, and enable complementary studies of eyeblink conditioning in human infants and developing rat pups (Stanton & Freeman, 1994). A first study (Ivkovich, Collins, Eckerman, Krasnegor, & Stanton, 1997) yielded only weak evidence of conditioning in infants given one session of paired training at either 4 or 5 months of age. A small subset of the infants receiving their first session at 4 months of age returned for a second paired training session 1 month later, however, and showed increased levels of conditioning that appeared to be the result of some aspect of their earlier training experience rather than developmental change. In the present study, we sought to replicate the finding of robust delay conditioning over two training sessions, but with training sessions only a week apart; establish whether the improved conditioning during the second session was the result of the retention of associative learning during the first session; and relate these findings to similar data obtained for rat pups (Stanton, Fox, & Carter, 1998). METHOD Subjects and Design Both 4- and 5-month-old infants (± 10 days) were recruited using local county birth records, and infants of both ages were randomly assigned to either a paired or an unpaired training group. Each group experienced two identical sessions of paired or unpaired training 6 to 8 days apart. Other 5-month-olds were assigned to one of two exposure-control groups. One of the control groups received unpaired presentations of the conditioned stimulus (CS) and unconditioned stimulus (US) during the first session (CS/US-exposure group). The other was exposed only to the training situation, without any CS or US presentations, during the first session (context-exposure group). Both these groups then received paired training 6 to 8 days later. Data are presented here for the 57 infants who completed a criterion number of trials during both sessions (at least 30 paired tone-airpuff trials during paired sessions or 30 tone-alone trials during unpaired sessions). 1 Procedures Each infant sat on a parent s lap facing a platform about 75 cm away. A standardized visual display of brightly colored objects was presented on the platform to attract and maintain the infant s attention. The parent held the infant in a way that gently restrained large motor movements and maintained roughly the same orientation throughout the session. A specially crafted soft band was secured, using Velcro, 1. An additional 54 infants (twenty 4-month-olds and thirty-four 5-montholds) were excluded from analyses because of failure to achieve the criterion number of trials (n = 25), rescheduling conflicts (n = 26), or technical difficulties (n = 3). Chi-square analyses on subjects with an insufficient number of trials revealed no evidence of selective attrition based on gender (χ 2 [1, N = 82] = 0.39), age (χ 2 [1, N = 82] = 0.50), or conditioning procedures (for Session 1: paired vs. unpaired, χ 2 [1, N = 59] = 2.96; paired vs. CS/US-exposure, χ 2 [1, N = 27] = 0.30; paired vs. context-exposure, χ 2 [1, N = 24] = 0.00). 4 Copyright 1999 American Psychological Society VOL. 10, NO. 1, JANUARY 1999

2 D. Ivkovich et al. across the infant s forehead and around the head to support a flexible plastic tube that delivered the airpuff US (approximately 1 /20 psi) to the subject s right eye. The airpuff was generated by an audio speaker that acted as a bellows device and ejected a momentary pulse of air. Two small 7-ohm speakers, 18 in. to either side of the infant s head, delivered the tone CS (1 khz, 80 db). Background instrumental music was played at low volume so that the pure-tone CS remained distinct. Two cameras, placed about 1 m to the front and right of the infant, yielded video records of the infant s head and of a signal box with a trial counter and lights indicating when the tone and airpuff stimuli were on. Electromyographic (EMG) records of eyeblinks were collected using three gel-pad electrodes positioned at the corner of the right eye, on the right temple, and at the back of the neck. A custom-built eyeblink conditioning system (U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina) controlled presentation of the stimuli and amplified and integrated EMG records for subsequent analysis. Paired training sessions A 750-ms tone was presented so that the last 100 ms overlapped and co-terminated with a gentle puff of air to the right eye. The intertrial interval varied from 8 to 16 s (average = 12 s). Every 6th trial in the 10- trial blocks, as well as Trials 1 and 2 at the start of the session, was an airpuff-alone trial to test for somatosensory responsiveness. Every 10th trial was a tone-alone trial to test for conditioned responding. The maximum number of trials presented was 50 (about 15 to 20 min), but the session was terminated earlier if the infant became overly fussy. Unpaired training sessions During unpaired training sessions, there was no associative contingency between the tone and airpuff. Subjects experienced, at most, the same 43 tones and 45 puffs as used for paired training, but the stimuli were presented explicitly unpaired, 4 to 8 s apart (average = 6 s) in a manner that matched the paired condition for stimulus density. The criteria for terminating a session were the same as those for paired sessions. Context-exposure sessions The context-exposure group received a training session that was identical to a paired training session except that no tone or airpuff stimuli were delivered. Response measures The primary measure of conditioning was the percentage of conditioned responses (CRs). For analysis purposes, trials were reblocked into sets of 6 trials, all of one trial type, for five successive blocks (total of 30 trials). For paired training sessions, the percentage of CRs was obtained for blocks of 6 paired trials; for unpaired sessions, the percentage of CRs was obtained for blocks of 6 tone-alone trials, with trials sampled to correspond in sequence with the tones on paired trials during paired sessions. Two independent observers coded the video records frame by frame for the occurrence and timing of a blink of the right eye during each trial. CRs were defined as blinks that started within 0 to 22 frames (367 ms) prior to onset of the airpuff. Unconditioned responses (URs) were blinks that started within 1 to 15 frames (250 ms) after onset of the airpuff. Trials in which the subject s right eye was out of view were excluded (< 10%). The percentage of agreement between observers was 98%. Because obtaining the EMG recordings was more problematic than coding the videotapes, the primary data presented here relied on video. EMG recordings were sometimes sensitive to facial and head movements and showed large intersubject variability in signal-to-noise ratio, but were usable for about 83% of the sessions. For these sessions, UR amplitudes on individual trials, confirmed by video analysis and excluding trials with a CR, were averaged across sessions within conditioning group. No reliable differences in UR amplitude that could produce any of the differences in conditioning reported here were obtained (see Table 1). RESULTS Conditioning at 4 and 5 Months of Age Results were analyzed separately at each age using between-within analyses of variance (ANOVA) with a significance level of.05. During the first training session, there was no increase in the percentage of CRs across blocks at either age and no differences between the paired and unpaired groups in the percentage of CRs (see Fig. 1). However, at both ages, substantial increases and group differences in the percentage of CRs emerged during the second session. A Group Session Block ANOVA at each age revealed main effects of group (F[1, 19] = 9.05, p <.01, for 4-month-olds; F[1, 19] = 9.23, p <.01, for 5-month-olds) and session (F[1, 19] = 17.09, p <.001, for 4- month-olds; F[1, 19] = 35.0, p <.001, for 5-month-olds). There were also interactions of group and block (F[4, 76] = 5.4, p <.001, for 4- month-olds; F[4, 76] = 3.45, p <.01, for 5-month-olds) and group and session (F[1, 19] = 10.0, p <.01, for 4-month-olds; F[1, 19] = 30.4, p <.01, for 5-month-olds). Figure 1 suggests that the percentage of CRs increased with age during Session 2, reaching around 80% for 5-month-olds, as compared with 60% for 4-month-olds. However, ANOVAs examining age differences for the paired groups either across sessions or across the first two blocks of Session 2 did not yield statistically significant effects involving age. Table 1. Mean amplitude of unconditioned responses as a function of age, group, and session Age (months) Session 1 Session 2 Paired training ± 0.8 (8) 3.8 ± 0.6 (7) ± 0.5 (10) 4.8 ± 0.5 (10) Unpaired training ± 0.8 (7) 4.6 ± 0.6 (8) ± 0.6 (8) 4.2 ± 0.8 (10) Conditioned stimulus/unconditioned stimulus exposure ± 0.7 (4) 5.8 ± 0.8 (6) Context exposure 5 Not applicable 5.6 ± 0.9 (7) Note. The numbers in parentheses show the numbers of subjects with usable electromyographic records. VOL. 10, NO. 1, JANUARY

3 Eyeblink Conditioning in Human Infants Fig 2. Mean (±SE) percentage of conditioned responses (CRs) as a function of trial block and session, for three groups of 5-month-olds. These groups underwent different experiences during Session 1, but all received paired training during Session 2. See the text for details. Fig. 1. Mean (±SE) percentage of conditioned responses (CRs) as a function of trial block and training session, for cross-sectional samples of 4- and 5-month-old infants in the paired and unpaired groups. Data are presented for six-trial blocks of paired trials (paired groups) or corresponding tone-alone trials (unpaired groups). Conditioning at 5 Months as a Function of Type of Prior Experience The 5-month paired group was compared with the two additional groups of 5-month-olds who received different experiences during their first session (see Fig. 2). For Session 1, a 3 (group) 5 (block) ANOVA did not yield any significant differences between groups. However, a similar ANOVA for Session 2 revealed significant main effects of group (F[2, 22] = 6.06, p <.01) and block (F[4, 88] = 10.90, p <.001), but no Group Block interaction. The paired group, which was undergoing its second session of paired training, showed a significantly higher percentage of CRs relative to the two groups that were experiencing paired training for the first time. Newman-Keuls tests revealed a significant difference between the paired group and each of the other groups (CS/US-exposure: p =.03; context-exposure: p =.01), which did not differ between themselves. It appears a significant Group Block interaction did not occur because the paired group started at around 50% CRs in the first block of the second session and maintained a higher percentage of CRs across the session, whereas the other groups started at around 10% CRs. Planned comparisons on this first block yielded a significant difference between the paired group and each of the other groups (CS/US-exposure: F[1, 22] = 7.55, p =.01; context-exposure: F[1, 22] = 8.83, p <.01). Also, a planned comparison of the last block of the first session and the first block of the second session showed a significant increase in percentage of CRs for the paired group only, F(1, 22) = 12.12, p <.01. DISCUSSION We have demonstrated robust delay eyeblink conditioning in 4- and 5-month-old human infants given two sessions of paired training. The levels of conditioning obtained were higher than those previously reported for younger human infants given similar amounts of training 6 VOL. 10, NO. 1, JANUARY 1999

4 D. Ivkovich et al. (Lintz, Fitzgerald, & Brackbill, 1967; Little et al., 1984; Naito & Lipsitt, 1969) but lower than those commonly observed in human adults, who reach conditioning levels of 60% to 80% CRs in less than 50 trials (Solomon & Pendlebury, 1988). It is hazardous to infer age differences in conditioning from comparisons across these studies because of the many procedural differences that distinguish them and the absence of detailed parametric data. For example, Little et al. (1984) found no delay conditioning in 10- to 30-day-old infants with the 500- ms interstimulus interval (ISI) that is optimal for adults (Kimble, 1947), but moderate levels of conditioning with an ISI of 1,500 ms. Nevertheless, the pattern of findings that emerges across studies is not inconsistent with the suggestion that delay eyeblink conditioning in human subjects develops gradually but substantially during the first 5 months of life. Substantial postnatal development of simple delay eyeblink conditioning already has been well established for rat pups (Freeman, Spencer, Skelton, & Stanton, 1993; Stanton et al., 1998; Stanton & Freeman, 1994; Stanton, Freeman, & Skelton, 1992). Human infants tested across two sessions in the present study were able to retain conditioning from one session to the next. Little et al. (1984) also observed retention of eyeblink conditioning over a 10-day period in human infants, starting around 20 days of age. However, retention in that study was not evident at the outset of retention testing but rather took the form of savings during subsequent training. In contrast, retention in the present study appeared as an increase in the percentage of CRs from the outset of retention testing for the groups that received paired training. This raises the possibility that long-term retention of eyeblink conditioning also improves during the first 5 months of life. The present findings also established that, even when there is little expression of conditioning during a first training session, there is a carryover effect on later conditioning that is not owing to nonassociative factors such as exposure to the context or the conditioning stimuli per se. A similar result has been observed in developing rats (Stanton et al., 1998). Three groups of rat pups received either paired, unpaired, or no training at 17 days of age; then, at 20 days of age, all groups received paired training. Little evidence of eyeblink conditioning was observed among the 17-day-old rats. However, paired training at this age produced substantial savings relative to the other two groups during the subsequent training at 20 days of age. These findings suggest that the distinction between acquisition and expression of learning may be important in developmental studies of Pavlovian conditioning, and they underscore the value of the three-group design (paired, CS/US-exposure, context-exposure) we employed for addressing this issue. It also is noteworthy that in the present study, prior unpaired training did not impair subsequent paired conditioning in 5-month-old infants. During Session 2, infants in the CS/US-exposure group did not perform worse than the infants in the context-exposure group, who were experiencing the CS and US for the first time. In adult subjects, preexposure to unpaired presentations of the CS and US impairs subsequent performance (relative to a naive group) during paired training, a phenomenon attributed to conditioned inhibition (Rescorla, 1967) or learned irrelevance (Mackintosh, 1974). There was no evidence for such an impairment in the present study or in a similar study involving infant rodents (Stanton et al., 1998). Infant mammals may not be able to learn that the CS and US are negatively correlated during unpaired training in the way that adults seem to do. According to one theoretical account of such learning (Rescorla, 1972), infants may not show this effect because of differences in attentional, contextual, or configural processing that may reflect immaturity of temporal cortical structures (Allen & Gluck, 1997; Nicolle, Barry, Veronesi, & Stanton, 1989; Rudy, 1992). In summary, these findings have established that the delay eyeblink conditioning procedures used are effective for 4- and 5-month-old human infants. These procedures can now be used to examine in more detail the normative ontogeny of eyeblink conditioning phenomena. Further, these findings have revealed phenomena (e.g., unexpressed learning and the absence of impairment following unpaired CS-US exposure) similar to those seen in the developing rat pup, illustrating the potential of complementary studies of eyeblink conditioning in human infants and animal models of early mammalian development. Acknowledgments We would like to thank Elizabeth Frederick, Aileen Haggerty, Jennie Lee, Betty Tao, Carrie Westlake, and Minna Wiley for their assistance with this work and Barbara Goldman for sharing with us her experience in infant eyeblink conditioning. Support for this research was provided in part by U.S. Environmental Protection Agency Cooperative Agreement No. CR and Social and Behavioral Sciences Research Grant No. 12-FY from the March of Dimes Foundation to C.O. Eckerman and by National Institute of Mental Health Postdoctoral Fellowship No. MH to D. Ivkovich. 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