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Fear Potentiation and Fear Inhibition in a Human Fear-Potentiated Startle Paradigm
Fear Potentiation and Fear Inhibition in a Human Fear-Potentiated Startle Paradigm Tanja Jovanovic, Megan Keyes, Ana Fiallos, Karyn M. Myers, Michael Davis, and Erica J. Duncan Background: The inability to suppress excessive fear or anxiety is a significant clinical problem. In the laboratory, extinction is a preferred method for the study of fear inhibition; however, in this paradigm the same stimulus causes both elicitation (excitation) and inhibition of fear, making it difficult to know whether an experimental manipulation that affects extinction does so by affecting one or both of these processes. For this reason, we sought to develop a behavioral procedure in humans that would render a stimulus primarily inhibitory. Methods: We adapted a conditional discrimination procedure (AX⫹/BX⫺), previously validated in animals, to a human fear-potentiated startle paradigm. Forty-one healthy volunteers were presented with one set of colored lights paired with the delivery of aversive airblasts to the throat (AX⫹) and a different series of lights presented without airblasts (BX⫺). Results: Participants exhibited fear potentiation to AX⫹, discrimination between AX⫹ and BX⫺, and transfer of fear inhibition to A in an AB compound test but not in an AC compound test. Conclusions: We believe this procedure will advance clinical research on fear disorders, such as posttraumatic stress disorder and phobias, by providing an effective and relatively independent measure of fear potentiation and fear inhibition.
Key Words: Startle reaction, fear, classical conditioning, electromyography, inhibition, conditional discrimination
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xcessive fear and anxiety, along with an inability to overcome these emotions, are some of the defining characteristics of many psychiatric disorders, such as phobias, panic disorder, and posttraumatic stress disorder (PTSD). Animal models of fear conditioning and fear inhibition provide useful tools for the study of these phenomena; therefore, it is essential to translate these models to human research. Our goal in the present study was to design a human fear conditioning paradigm based on an animal model that can assess fear potentiation and fear inhibition under conditions in which they are relatively independent, using a fear-potentiated startle paradigm. Fear-potentiated startle is defined by the relative increase in the amplitude of the acoustic startle reflex when elicited in the presence of a conditioned stimulus (CS) previously paired with an aversive stimulus (unconditioned stimulus, US). Fear-potentiated startle can be demonstrated in animals and humans (Ameli et al 2001; Davis 1992; Grillon and Davis 1997; for a recent review see Grillon and Baas 2003). As a result, it provides an objective measure of the fear response and is an ideal paradigm for translational research (Davis et al 1993). In the laboratory, inhibition of fear traditionally has been studied with the use of two conditioning paradigms: extinction and conditioned inhibition. Each of these procedures, however, has certain disadvantages that can make it difficult to differentiate clearly the degree to which a given stimulus produces fear (excitation) versus inhibition of fear (inhibition). In the typical extinction paradigm, the CS is presented repeatedly in the
From the Emory University School of Medicine (TJ, AF, KMM, MD, EJD), Atlanta; Center for Behavioral Neuroscience (TJ, MK, AF, KMM, MD, EJD), Atlanta; Atlanta Veterans Affairs Medical Center (TJ, MK, AF, EJD), Decatur, Georgia; and Massachusetts Institute of Technology (AF), Cambridge, Massachusetts. Address reprint requests to Tanja Jovanovic, Ph.D., Atlanta Veterans Affairs Medical Center, Mental Health Service/116A, 1670 Clairmont Road, Decatur, GA 30033; E-mail: [email protected]. Received October 4, 2004; revised January 12, 2005; accepted February 18, 2005.
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absence of the US after CS–US pairings, with a resultant decrease in the measured level of fear to the CS. A great deal of evidence indicates that, after extinction, fear to the CS is not erased, but instead the CS now also engages a parallel inhibitory process, which competes with or suppresses fear elicited by that same CS (see Myers and Davis 2002). Because the CS now has both excitatory and inhibitory properties, it is difficult to tease apart whether a given experimental manipulation that affects extinction does so by affecting inhibition or excitation. In the traditional conditioned inhibition paradigm, one stimulus (A) is paired with an aversive US when presented in isolation (A⫹) and is not paired with the US when presented in compound with a second stimulus, B (BA⫺). After training on this A⫹, BA⫺ discrimination, A elicits a fear response, but BA does not. It can be demonstrated that the absence of fear to BA is due to inhibitory fear learning to B that counteracts the fear that otherwise would be elicited by A. The inhibitory impact of B can be measured independently of its effects on A by two means: 1) a retardation in the rate at which fear develops to B when it is subsequently paired with shock (a “retardation test”); and 2) its ability to reduce fear to a separately trained excitatory stimulus (a “summation test”) (Rescorla 1969). Thus, theoretically, conditioned inhibition training results in the separation of excitatory and inhibitory tendencies in different CSs. In practice, however, this outcome is not guaranteed. Interestingly, the A⫹, AB⫺ procedure is identical to the training protocol used in studies of second-order conditioning, in which A acts as a US to condition fear to B (Rescorla 1980). Although it is generally assumed that B initially becomes excitatory and then becomes inhibitory with further training on the A⫹, BA⫺ discrimination, one can show by using special test procedures that B can both be excitatory and inhibitory at the same time (see Falls and Davis 1997). As with extinction, then, in which a single stimulus both elicits and inhibits fear, this makes a clear separation of excitation and inhibition difficult. Human participants might be especially susceptible to second-order conditioning, as indicated by a study by Grillon and Ameli (2001), in which highand low-anxiety subjects trained on the A⫹, XA⫺ discrimination did not inhibit the fear response when X was paired with another previously reinforced stimulus B⫹. Myers and Davis (2004) have recently developed a discrimination procedure in rats that minimizes these problems and BIOL PSYCHIATRY 2005;57:1559 –1564 © 2005 Society of Biological Psychiatry
1560 BIOL PSYCHIATRY 2005;57:1559 –1564 allows for a more independent evaluation of excitation and inhibition of fear. The procedure, referred to as a conditional discrimination and abbreviated as AX⫹/BX⫺, is based on a paradigm used for other reasons in earlier learning theory experiments (Wagner and Rescorla 1972; Wagner et al 1968). In this experiment, the response to a third stimulus, X, is conditional upon the presence of either A or B. A becomes excitatory with training as the subject learns that A and X presented together predict the US. B becomes inhibitory in that B presented with X predicts safety from the US. In a critical subsequent test trial, presentation of A and B together (AB) results in a reduced fear response compared with the response to A. Separate experiments demonstrated that this decrease in responding to A in the presence of B was not due to external inhibition, which refers to an unconditioned decrease in conditioned responding to a well-trained CS when that CS is compounded with a novel stimulus (Pavlov 1927). External inhibition is believed to be an attentional effect in which the novel stimulus distracts the subject and prevents full processing of the CS. The AX⫹/BX⫺ discrimination also has the advantage of decreasing the problem posed by second-order conditioning because X, being only weakly excitatory, is far from an optimal first-order CS for second-order conditioning of B. In the present article, we report an adaptation of this AX⫹/ BX⫺ discrimination procedure to a human fear-potentiated startle paradigm. Forty-one healthy volunteers were presented with one set of colored lights paired with the delivery of aversive airblasts to the throat (AX⫹) and a different series of lights presented without airblasts (BX⫺). As opposed to the rodent paradigm, we only used compound stimuli in the experiment because we were concerned that people might see single cues as categorically different from two cues. One of the difficulties in translating animal paradigms to human participants is that humans tend to perceive compound stimuli as a unique, single stimulus rather than as a collection of separate stimulus elements (Williams et al 1995). Such configural processing would allow humans to solve the AX⫹, BX⫺ discrimination by treating AX as one stimulus and BX as another. As a result, they would not learn that stimulus B signaled safety but rather that stimulus BX did, making it unlikely that B would inhibit A in an AB test trial. In an effort to encourage subjects to process each light in the experiment as separate elements, in this study a response keypad was used during training to assess contingency awareness for lights A, B, and X separately, defined as the subject’s knowledge of the reinforcement contingencies in the experiment (Lovibond and Shanks 2002). This was accomplished by having the subjects rate each light as reinforced (threat) or non-reinforced (safe) by pressing different buttons on the keypad. In addition to encouraging subjects to consider the independent reinforcement value of each stimulus, the keypad was used to identify the subjects who were or were not aware of the reinforcement contingencies because this has been found to affect the magnitude of fear-potentiated startle. Thus Grillon (2002) found in a CS⫹, CS⫺ discrimination paradigm that subjects who were aware of the contingencies had approximately 70% fear-potentiated startle, whereas those who were not aware had only approximately 20%, as well as higher startle amplitudes on CS⫺ test trials. On the basis of the rodent model (Myers and Davis 2004), we hypothesized that startle to AB would be lower than startle to AX. As mentioned above, the human paradigm differed from the rodent protocol in that we did not have any single cues; therefore, we could not compare AB with A directly, but rather www.sobp.org/journal
T. Jovanovic et al
Figure 1. Diagram of the AX⫹/BX⫺ startle session. NA, noise alone.
AB with AX to assess fear inhibition. Finally, to demonstrate that a safety signal (B) would be more effective at inhibiting fear than the presentation of a novel stimulus (C), we predicted that startle should be lower on AB versus AC test trials.
Methods and Materials Participants Forty-one healthy subjects participated in the study after signing a consent form approved by the Emory University Institutional Review Board as indication of their informed consent. The sample included 16 women and 25 men, who ranged in age from 20 to 74 years. The subjects had no current or lifetime Axis I disorders, including substance abuse and dependence, as ascertained by the Structured Clinical Interview for the DSM-IV Axis I Disorders (First et al 1998) interview. All subjects were screened for auditory or visual impairment. Using an audiometer (Model MA27, Maico, Minneapolis, Minnesota) the subjects detected tones at a 30-dBA sound pressure level at frequencies ranging from 250 Hz to 4000 Hz. The subjects were not colorblind and had at least 20/40 vision in both eyes (with correction, if necessary) at day of testing. In addition, all subjects had negative results on urine toxicology screen. Startle Procedure The acoustic startle response (eyeblink component) was measured by electromyography (EMG) of the right orbicularis oculi muscle. Two 5-mm Ag/AgCl electrodes filled with electrolyte gel were positioned approximately 1 cm under the pupil and 1 cm below the lateral canthus, and a ground electrode was placed behind the right ear over the mastoid. All resistances were less than 6 K⍀. Electromyographic activity was amplified and digitized with a computerized EMG startle response monitoring system (SR-LAB; San Diego Instruments, San Diego, Caifornia). The EMG signal was filtered with low- and high-frequency cutoffs at 30 Hz and 1000 Hz, respectively. The system was set to record 250 1-msec readings starting at the onset of the startle stimulus. Subjects were seated and asked to look at the set of four lights mounted on the wall approximately 5 feet from their seat. All acoustic stimuli were delivered binaurally through headphones (model TDH-39-P; Maico, Minneapolis, Minnesota). The startle session (see diagram in Figure 1) began with a 1-min acclimation period consisting of 70-dBA broadband noise, which continued as the background noise throughout the session. The startle probe (noise burst) was either a 104- or 108-dBA sound pressure level, 40-msec burst of broadband noise with a near instantaneous rise time. According to methods established by Grillon and Davis (1997), the aversive stimulus (US) was a
T. Jovanovic et al
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Figure 2. Diagram of the trials in the AX⫹/BX⫺ session. The AB and AC trials were structured the same as the BX⫺ trials. NA, noise alone.
100-msec airblast with an intensity of 140 psi directed to the larynx, emitted by a compressed air tank attached to polyethylene tubing and controlled by a solenoid valve. A, B, C, and X were green, purple, orange, or blue lights ranging in light transmission from 4.0% to 4.2% (counterbalanced color assignment across subjects). Figure 1 shows a diagram of the AX⫹/ BX⫺ startle session. The test session began with a habituation phase consisting of six startle probes (three at 104 dB and three at 108 dB) to reduce initial startle reactivity and rule out non-startlers. To minimize individual variability in baseline startle, subjects were either assigned to the 104-dB session or the 108-dB session, according to startle level in the habituation phase (if startle was below 100 machine units, subjects were assigned to 108 dB). The conditioning phase included six startle probes presented alone, six trials in which stimuli A and X were paired with the US (AX⫹), and six trials in which stimuli B and X were not paired with the US (BX⫺) (see Figure 1). The AX⫹ stimuli were presented serially within a trial, and the order of A and X alternated randomly across trials. In the AX⫹, the first light came on and stayed on for 7 sec; after 3 sec, the second light came on, so that for the last 4 sec the two lights were presented together. The startle probe was presented at the end of 6 sec (when the two lights had been presented together for 3 sec) and was followed by the airblast 500 msec later (see trial diagram in Figure 2). The airblast lasted for 250 msec, and the lights stayed on for another 250 msec after that, so that both lights were still on during both the startle probe and the airblast. In the BX⫺ trials, as well as in the AB and AC test trials, there was no airblast; therefore, the first light stayed on for 6 sec and 250 msec, and the second light was presented for the last 3250 msec. In these trials as well, the startle probe was presented at the end of the first 6 sec, and the lights stayed on for another 250 msec after the startle probe. The testing phase consisted of two blocks: each block included six startle probes presented alone and six presentations of AB or AC, respectively (see Figure 1). The order of the two blocks (i.e., one with AB trials and one with AC trials) was counterbalanced across subjects. In between the two blocks, there was a brief reconditioning phase in which AX⫹ was presented with the US again to maintain fear potentiation to AX. We did not repeat the presentations of BX because these trials were not reinforced and therefore habituation to the US was not a concern, whereas prolonging the testing session more than necessary would have raised the issue of startle habituation. In all phases of the experiment, intertrial intervals were of randomized duration, ranging from 9 to 22 sec. Response Keypad A response keypad unit (SuperLab; Cedrus, San Pedro, California) was incorporated into the startle session so that the EMG startle response monitoring system (SR-LAB) signaled the onset
of a light in the SuperLab software program. Each trial contained two light components (e.g., A and X). Subjects were instructed to respond to each light separately on each trial by pressing one of three buttons: one when they expected a light to be followed by the airblast, a second button when they did not expect the light to be followed by the airblast, and a third button when they were uncertain of what to expect. Awareness We assessed awareness of the experimental contingency according to the subjects’ keypad responses. First, the subjects had to have two consecutive correct responses to each of the two types of training trials (AX⫹ and BX⫺). The second criterion was to correctly label the last training trial (i.e., trial number 6) of each type. We operationally defined correct responses to AX⫹ trials as expectations of an airblast when presented with the A light as well as during an X light when it followed A; the correct responses to BX⫺ were expectations of no airblast on the B light as well as on an X light when it followed B. Subjects who did not meet both criteria were defined as unaware and excluded from further analyses. Statistical Analysis Repeated-measures analysis of variance (ANOVA) models were used to test effects of trial type (four levels: AX, BX, AB, and AC). The dependent variable was the difference score calculated by subtracting the startle amplitude during light presentation from the baseline startle amplitude, in this case the mean of the startle amplitudes on noise-alone trials (probes delivered in the absence of the light) during the conditioning and testing phase. Because of the variable nature of the startle response, we did not want to use a single data point for each individual and thus averaged three trials of each trial type. For AX⫹ and BX⫺ trial types we wanted to capture the results of training and therefore we calculated the mean of the last three trials of each type; for AB and AC trial types we wanted to test immediate transfer of safety (without the effects of learning) and thus used only the first three trials of each type. Furthermore, to account for habituation to the US throughout the session we calculated a mean AX, which included the re-conditioning block of AX, and subtracted the AB trial types and the AC trial types from the mean AX (AXAVG) to measure inhibition. Finally, to determine whether subjects transferred safety on the very first presentation of AB, we tested the linear effect of the mean AX and each of the first three trials of either AB or AC. To correct for violations of the sphericity assumption in the repeated-measures ANOVAs, we used Huynh-Feldt ε adjustments. Significant main effects of trial type were analyzed with specific contrast comparing AX with BX, AB with AX, AC with AX, and AB with AC. We also tested the four trial types for a www.sobp.org/journal
1562 BIOL PSYCHIATRY 2005;57:1559 –1564
T. Jovanovic et al was not [F (1,25) ⬍ 1, 2 ⫽ .02]. The data were also analyzed with order of testing (AB first vs. AC first) and decibel level (104 vs. 108) as between-groups variables. Neither the order of the testing blocks nor the decibel level interacted with the results. Conditioned Versus External Inhibition We looked at inhibition by subtracting the average startle amplitude on the first three AB or AC trial types from AXAVG (AX averaged between the second block of conditioning and the re-conditioning block to take into account habituation to the US, as described in Statistical Analysis). In this analysis, AB trials showed significantly more inhibition than the AC trials [F (1,25) ⫽ 4.79, p ⬍ .05, 2 ⫽ .16) (see Figure 4).
Figure 3. Startle potentiation to AX, BX, AB, and AC according to the difference score, calculated as the difference in startle to the acoustic probe in the presence and absence of the light cue.
quadratic trend. Effect sizes of the individual effects are reported with partial 2. All analyses were conducted with SPSS 10.0 for Macintosh (SPSS, Chicago, Illinois).
Results Of the 41 subjects who signed informed consent, 14 were excluded from further analyses because they were unaware of the experimental contingencies as measured by the response keypad (see Awareness section in Methods and Materials), and one was excluded owing to data collection error. This resulted in a final sample size of 26 subjects; 9 were female and 17 were male. Of the 26 subjects, 19 used the keypad as directed, pressing a button for each light; one female and six male subjects only pressed a button once per trial when the two lights were presented together. The testing blocks were evenly counterbalanced across subjects, with 13 receiving the AB testing block first, and 13 receiving the AC block first. Fear-Potentiated Startle Startle was robustly potentiated in the presence of all trial types, as indicated by the intercept term of the ANOVA; that is, the difference score between the trials with lights and the noise-alone trials was significantly different from zero [F (1,25) ⫽ 19.80, p ⬍ .001, 2 ⫽ .44]. Furthermore, there was an overall significant effect of trial type on the difference score [F (3,75) ⫽ 4.96, p ⬍ .01, 2 ⫽ .17]. We analyzed the specific trial type contrasts of AX versus BX, AX versus AB, AX versus AC, and AC versus AB. In the second block of conditioning we found significantly larger startle potentiation to the reinforced trials (AX) than to the non-reinforced trials (BX) [F (1,25) ⫽ 7.94, p ⬍ .01, 2 ⫽ .24] (see Figure 3). We also found significantly less potentiation to AB compared with AX [F (1,25) ⫽ 7.88, p ⬍ .01, 2 ⫽ .24], whereas there was no decreased potentiation to AC compared with AX. Comparing AB and AC directly, we found that AB was potentiated significantly less than AC [F (1,25) ⫽ 4.79, p ⬍ .05, 2 ⫽ .16] (Figure 3). We found that the subjects tended to habituate to the US throughout the session, so that startle potentiation to the second block of conditioning AX was slightly larger than to the reconditioning block of AX [F (1,25) ⫽ 3.29, p ⬍ .08, 2 ⫽ .12]. Thus, we decided to average these two values of AX (AXAVG) and test whether this more conservative analysis would also show AB inhibition. We again found that startle to AB was smaller than AXAVG [F (1,25) ⫽ 6.34, p ⬍ .05, 2 ⫽ .20], whereas startle to AC www.sobp.org/journal
Configural Versus Elemental Processing Transfer of inhibition of fear-potentiated startle to the AB trials suggests that the A and B lights were processed as elements rather than as configural stimuli; that is, A, rather than an A-and-X compound, was the excitatory stimulus, whereas B was the inhibitory stimulus. The data from the response pad indicate that this was the case: on the last training trial, A (in an AX trial) was labeled as reinforced by 89.5% of the subjects, whereas B (in a BX trial) was always labeled as nonreinforced [2(6) ⫽ 66.9, p ⬍ .001]. On the first testing trial, B (in an AB trial) was labeled as nonreinforced 94.7% of the time, whereas C (in an AC trial) was labeled as unknown 68.4% of the time. In addition, as further indication of transference, the first presentation of A in the testing trial (an AB trial, where B preceded A) was only labeled as reinforced 10.5% of the time, compared with 89.5% at the end of training [2(6) ⫽ 33.2, p ⬍ .001].
Discussion In this study, we sought to develop a new method of measuring inhibition of fear in humans that might reduce some of the disadvantages of the more commonly used methods, such as extinction and conditioned inhibition. To do this, we adapted a conditional discrimination procedure, of the form AX⫹/BX⫺, developed to measure fear inhibition in rats with the fearpotentiated startle paradigm (Myers and Davis 2004). Using lights of four different colors (A, B, C, X), we paired colors A and X with a blast of air to the throat, whereas colors B and X signaled that no shock would occur. To encourage subjects to use elemental rather than configural strategies during conditioning
Figure 4. Inhibition of potentiated startle to AB and AC according to a difference score from AXAVG; that is, subtracting the startle amplitude to AB (or AC) from the startle amplitude for AXAVG (the AX value was averaged between conditioning and reconditioning to account for habituation).
T. Jovanovic et al (Williams et al 1995), the subjects used a response keypad to label the reinforcement contingencies to each light in a trial as “safe,” “danger,” or “do not know,” so that A would be perceived as the danger signal and B would be perceived as the safety signal. The response keypad also allowed us to determine whether the subjects were learning the discrimination task on a cognitive level (i.e., whether they were aware of the contingencies in the experiment). We found that approximately 34% of the subjects were not aware of the contingencies in the experiment, according to their keypad performance, and excluded them from further analyses. Using potentiation of the eyeblink component of startle as our operational definition of fear, we found that the aware subjects startled more to AX than to noise alone and startled less to AB than to AX, indicating that they were both potentiating to the fear stimulus and inhibiting the fear response in the presence of safety signals. To demonstrate that B was indeed a safety cue, we had to show that subjects could immediately transfer safety on a subsequent AB test trial; that is, that the decrement in startle to AB relative to AX was not an effect of learning that AB was non-reinforced. We tested this by focusing on the first three presentations of AB and found that the subjects did indeed show immediate transfer on those trials. Furthermore, the response pad data indicate that the subjects were transferring safety on a cognitive level as well: on the very first presentation of AB, the subjects immediately recognized B as a safety signal and dramatically reduced the level of danger expected with A when it was paired with B. This represents the first study to demonstrate transfer in a conditioned inhibition experiment with fear-potentiated startle in humans. In the Grillon and Ameli (2001) study, in which a conditioned inhibition paradigm of the form A⫹, XA⫺ was used, no transfer of the safety cue (X) to a separately trained danger cue (B) was found, even when the investigators looked at as many as three blocks of B preceded by X. There are two possible reasons for the difference between Grillon’s study and our study. First, our experimental paradigm was designed to minimize second-order conditioning to the safety cue because B was never actually paired with A but instead was paired with X, which was shown in the rodent study to be much less fearful than A (Myers and Davis 2004); however, because Grillon found significant differences between the danger stimulus (A⫹) and the conditioned inhibitor (XA⫺), it is unlikely that X was second-order conditioned by A. The lack of transfer in the Grillon study could also be explained by configural processing, such that the AX compound trials were perceived by the subjects as a unique, new stimulus rather than as a combination of previously learned elements. Although Grillon used serial presentations of the stimuli to minimize configuration, that approach might not have been sufficient to induce elemental processing. In our study, startle to the combination of danger and safety (AB) was more inhibited than startle to the combination of danger and novelty (AC). We believe that the use of the response pad forced the subjects to perceive the stimuli as elements, which resulted in the transfer of safety and the subsequent inhibition of fear in our subjects. In addition, prior experience with compound stimuli seems to reduce external inhibition (Myers and Davis 2004), which might be another reason that we saw little external inhibition in the current study. Although we believe this AX⫹/BX⫺ design is superior to the typical A⫹, XA⫺ design, it still has the usual problems encountered with the use of fear conditioning in humans; namely, habituation to the aversive stimulus and a large proportion of
BIOL PSYCHIATRY 2005;57:1559 –1564 1563 unaware subjects (Grillon and Baas 2003). The problem of habituation can be remedied somewhat statistically; however, the unawareness issue requires testing larger sample sizes to replace subjects who do not learn the experimental contingencies. The AX⫹/BX⫺ paradigm allows for a more independent analysis of fear potentiation and fear inhibition and can be used in clinical populations to determine which of the two processes is dysregulated. For instance, PTSD patients might have normal levels of fear potentiation but have trouble inhibiting fear responses (Grillon and Morgan 1999). One of the advantages of the current study was the use of an airblast as the aversive stimulus, which is well tolerated by psychiatric patients and allows for larger group differences when comparing patients with control subjects (Grillon and Baas 2003). In future studies, this startle experiment can be used to test such patients, as well as to assess psychopharmacologic treatments to determine how they affect each process independently. In summary, we found that the AX⫹/BX⫺ paradigm, adapted from the animal model of conditional discrimination, can measure both fear potentiation and fear inhibition in human subjects under conditions in which the normal problems of second-order conditioning, configural learning, and external inhibition are minimized.
This research was supported by the Mental Health Service, Atlanta Department of Veterans Affairs Medical Center; the Science and Technology Center Program, the Center for Behavioral Neuroscience, of the National Science Foundation under Agreement No. IBN-9876754 (Venture grant, EJD); the American Psychiatric Association/Glaxo SmithKline (EJD); National Institute of Mental Health grants 1R24MH067314-01A1 (B. Rothbaum), R37 MH47840 (MD); Kirschstein National Research Service Award Individual Fellowship 1F32 MH070129-01A2 (TJ); and the Woodruff Foundation, Emory University School of Medicine. We gratefully acknowledge the input of Barbara Rothbaum, Kerry Ressler, Kim Huhman, Stephan Hamann, and Christian Grillon. Ameli R, Ip C, Grillon C (2001): Contextual fear-potentiated startle conditioning in humans: Replication and extension. Psychophysiology 38:383–390. Davis M (1992): The role of the amygdala in fear and anxiety. Ann Rev Neurosci 15:353–375. Davis M, Falls WA, Campeau S, Kim M (1993): Fear-potentiated startle: A neural and pharmacological analysis. Behav Brain Res 58:175–198. Falls WA, Davis M (1997): Inhibition of fear-potentiated startle can be detected after the offset of a feature trained in a serial feature negative discrimination. J Exp Psychol Anim Behav Process 23:3–14. First MB, Spitzer RL, Gibbon M, Williams JBW (1998): Structured Clinical Interview for DSM-IV Axis I Disorders-Patient Edition (SCID-I/P, Version 2.0, 8/98 revision). New York: New York State Psychiatric Institute. Grillon C (2002): Associative learning deficits increase symptoms of anxiety in humans. Biol Psychiatry 51:851– 858. Grillon C, Ameli R (2001): Conditioned inhibition of fear-potentiated startle and skin conductance in humans. Psychophysiology 38:807– 815. Grillon C, Baas J (2003): A review of the modulation of the startle reflex by affective states and its application in psychiatry. Clin Neurophysiol 114: 1557–1579. Grillon C, Davis M (1997): Fear-potentiated startle conditioning in humans: Effects of explicit and contextual cue conditioning following paired vs. unpaired training. Psychophysiology 34:451– 458. Grillon C, Morgan CA III (1999): Fear-potentiated startle conditioning to explicit and contextual cues in Gulf War veterans with posttraumatic stress disorder. J Abnorm Psychol 108:134 –142.
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1564 BIOL PSYCHIATRY 2005;57:1559 –1564 Lovibond PF, Shanks DR (2002): The role of awareness in Pavlovian conditioning: Empirical evidence and theoretical implications. J Exp Psychol Anim Behav Proces 28:3–26. Myers KM, Davis M (2002): Behavioral and neural analysis of extinction: A review. Neuron 36:567–584. Myers KM, Davis M (2004): AX⫹, BX⫺ discrimination learning in the fearpotentiated startle paradigm: Possible relevance to inhibitory fear learning in extinction. Learn Mem 11:464 – 475. Pavlov IP (1927): Conditioned Reflexes [Anrep GV, translator]. London: Oxford University Press.
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T. Jovanovic et al Rescorla RA (1969): Pavlovian conditioned inhibition. Psychol Bull 72:77–94. Rescorla RA (1980): Pavlovian Second-Order Conditioning: Studies in Associative Learning. Hillsdale, New Jersey: Erlbaum. Wagner AR, Logan FA, Haberlandt K, Price T (1968): Stimulus selection in animal discrimination learning. J Exp Psychol 76:171–180. Wagner AR, Rescorla RA (1972): Inhibition and learning. In: Boakes RA, Halliday MS, editors. Inhibition in Pavlovian Conditioning. London: Academic Press. Williams DA, Sagness KE, McPhee JE (1995): Configural and elemental strategies in predictive learning. J Exp Psychol Learn Mem Cogn 20:694 –709.
Key Words: Startle reaction, fear, classical conditioning, electromyography, inhibition, conditional discrimination
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xcessive fear and anxiety, along with an inability to overcome these emotions, are some of the defining characteristics of many psychiatric disorders, such as phobias, panic disorder, and posttraumatic stress disorder (PTSD). Animal models of fear conditioning and fear inhibition provide useful tools for the study of these phenomena; therefore, it is essential to translate these models to human research. Our goal in the present study was to design a human fear conditioning paradigm based on an animal model that can assess fear potentiation and fear inhibition under conditions in which they are relatively independent, using a fear-potentiated startle paradigm. Fear-potentiated startle is defined by the relative increase in the amplitude of the acoustic startle reflex when elicited in the presence of a conditioned stimulus (CS) previously paired with an aversive stimulus (unconditioned stimulus, US). Fear-potentiated startle can be demonstrated in animals and humans (Ameli et al 2001; Davis 1992; Grillon and Davis 1997; for a recent review see Grillon and Baas 2003). As a result, it provides an objective measure of the fear response and is an ideal paradigm for translational research (Davis et al 1993). In the laboratory, inhibition of fear traditionally has been studied with the use of two conditioning paradigms: extinction and conditioned inhibition. Each of these procedures, however, has certain disadvantages that can make it difficult to differentiate clearly the degree to which a given stimulus produces fear (excitation) versus inhibition of fear (inhibition). In the typical extinction paradigm, the CS is presented repeatedly in the
From the Emory University School of Medicine (TJ, AF, KMM, MD, EJD), Atlanta; Center for Behavioral Neuroscience (TJ, MK, AF, KMM, MD, EJD), Atlanta; Atlanta Veterans Affairs Medical Center (TJ, MK, AF, EJD), Decatur, Georgia; and Massachusetts Institute of Technology (AF), Cambridge, Massachusetts. Address reprint requests to Tanja Jovanovic, Ph.D., Atlanta Veterans Affairs Medical Center, Mental Health Service/116A, 1670 Clairmont Road, Decatur, GA 30033; E-mail: [email protected]. Received October 4, 2004; revised January 12, 2005; accepted February 18, 2005.
0006-3223/05/$30.00 doi:10.1016/j.biopsych.2005.02.025
absence of the US after CS–US pairings, with a resultant decrease in the measured level of fear to the CS. A great deal of evidence indicates that, after extinction, fear to the CS is not erased, but instead the CS now also engages a parallel inhibitory process, which competes with or suppresses fear elicited by that same CS (see Myers and Davis 2002). Because the CS now has both excitatory and inhibitory properties, it is difficult to tease apart whether a given experimental manipulation that affects extinction does so by affecting inhibition or excitation. In the traditional conditioned inhibition paradigm, one stimulus (A) is paired with an aversive US when presented in isolation (A⫹) and is not paired with the US when presented in compound with a second stimulus, B (BA⫺). After training on this A⫹, BA⫺ discrimination, A elicits a fear response, but BA does not. It can be demonstrated that the absence of fear to BA is due to inhibitory fear learning to B that counteracts the fear that otherwise would be elicited by A. The inhibitory impact of B can be measured independently of its effects on A by two means: 1) a retardation in the rate at which fear develops to B when it is subsequently paired with shock (a “retardation test”); and 2) its ability to reduce fear to a separately trained excitatory stimulus (a “summation test”) (Rescorla 1969). Thus, theoretically, conditioned inhibition training results in the separation of excitatory and inhibitory tendencies in different CSs. In practice, however, this outcome is not guaranteed. Interestingly, the A⫹, AB⫺ procedure is identical to the training protocol used in studies of second-order conditioning, in which A acts as a US to condition fear to B (Rescorla 1980). Although it is generally assumed that B initially becomes excitatory and then becomes inhibitory with further training on the A⫹, BA⫺ discrimination, one can show by using special test procedures that B can both be excitatory and inhibitory at the same time (see Falls and Davis 1997). As with extinction, then, in which a single stimulus both elicits and inhibits fear, this makes a clear separation of excitation and inhibition difficult. Human participants might be especially susceptible to second-order conditioning, as indicated by a study by Grillon and Ameli (2001), in which highand low-anxiety subjects trained on the A⫹, XA⫺ discrimination did not inhibit the fear response when X was paired with another previously reinforced stimulus B⫹. Myers and Davis (2004) have recently developed a discrimination procedure in rats that minimizes these problems and BIOL PSYCHIATRY 2005;57:1559 –1564 © 2005 Society of Biological Psychiatry
1560 BIOL PSYCHIATRY 2005;57:1559 –1564 allows for a more independent evaluation of excitation and inhibition of fear. The procedure, referred to as a conditional discrimination and abbreviated as AX⫹/BX⫺, is based on a paradigm used for other reasons in earlier learning theory experiments (Wagner and Rescorla 1972; Wagner et al 1968). In this experiment, the response to a third stimulus, X, is conditional upon the presence of either A or B. A becomes excitatory with training as the subject learns that A and X presented together predict the US. B becomes inhibitory in that B presented with X predicts safety from the US. In a critical subsequent test trial, presentation of A and B together (AB) results in a reduced fear response compared with the response to A. Separate experiments demonstrated that this decrease in responding to A in the presence of B was not due to external inhibition, which refers to an unconditioned decrease in conditioned responding to a well-trained CS when that CS is compounded with a novel stimulus (Pavlov 1927). External inhibition is believed to be an attentional effect in which the novel stimulus distracts the subject and prevents full processing of the CS. The AX⫹/BX⫺ discrimination also has the advantage of decreasing the problem posed by second-order conditioning because X, being only weakly excitatory, is far from an optimal first-order CS for second-order conditioning of B. In the present article, we report an adaptation of this AX⫹/ BX⫺ discrimination procedure to a human fear-potentiated startle paradigm. Forty-one healthy volunteers were presented with one set of colored lights paired with the delivery of aversive airblasts to the throat (AX⫹) and a different series of lights presented without airblasts (BX⫺). As opposed to the rodent paradigm, we only used compound stimuli in the experiment because we were concerned that people might see single cues as categorically different from two cues. One of the difficulties in translating animal paradigms to human participants is that humans tend to perceive compound stimuli as a unique, single stimulus rather than as a collection of separate stimulus elements (Williams et al 1995). Such configural processing would allow humans to solve the AX⫹, BX⫺ discrimination by treating AX as one stimulus and BX as another. As a result, they would not learn that stimulus B signaled safety but rather that stimulus BX did, making it unlikely that B would inhibit A in an AB test trial. In an effort to encourage subjects to process each light in the experiment as separate elements, in this study a response keypad was used during training to assess contingency awareness for lights A, B, and X separately, defined as the subject’s knowledge of the reinforcement contingencies in the experiment (Lovibond and Shanks 2002). This was accomplished by having the subjects rate each light as reinforced (threat) or non-reinforced (safe) by pressing different buttons on the keypad. In addition to encouraging subjects to consider the independent reinforcement value of each stimulus, the keypad was used to identify the subjects who were or were not aware of the reinforcement contingencies because this has been found to affect the magnitude of fear-potentiated startle. Thus Grillon (2002) found in a CS⫹, CS⫺ discrimination paradigm that subjects who were aware of the contingencies had approximately 70% fear-potentiated startle, whereas those who were not aware had only approximately 20%, as well as higher startle amplitudes on CS⫺ test trials. On the basis of the rodent model (Myers and Davis 2004), we hypothesized that startle to AB would be lower than startle to AX. As mentioned above, the human paradigm differed from the rodent protocol in that we did not have any single cues; therefore, we could not compare AB with A directly, but rather www.sobp.org/journal
T. Jovanovic et al
Figure 1. Diagram of the AX⫹/BX⫺ startle session. NA, noise alone.
AB with AX to assess fear inhibition. Finally, to demonstrate that a safety signal (B) would be more effective at inhibiting fear than the presentation of a novel stimulus (C), we predicted that startle should be lower on AB versus AC test trials.
Methods and Materials Participants Forty-one healthy subjects participated in the study after signing a consent form approved by the Emory University Institutional Review Board as indication of their informed consent. The sample included 16 women and 25 men, who ranged in age from 20 to 74 years. The subjects had no current or lifetime Axis I disorders, including substance abuse and dependence, as ascertained by the Structured Clinical Interview for the DSM-IV Axis I Disorders (First et al 1998) interview. All subjects were screened for auditory or visual impairment. Using an audiometer (Model MA27, Maico, Minneapolis, Minnesota) the subjects detected tones at a 30-dBA sound pressure level at frequencies ranging from 250 Hz to 4000 Hz. The subjects were not colorblind and had at least 20/40 vision in both eyes (with correction, if necessary) at day of testing. In addition, all subjects had negative results on urine toxicology screen. Startle Procedure The acoustic startle response (eyeblink component) was measured by electromyography (EMG) of the right orbicularis oculi muscle. Two 5-mm Ag/AgCl electrodes filled with electrolyte gel were positioned approximately 1 cm under the pupil and 1 cm below the lateral canthus, and a ground electrode was placed behind the right ear over the mastoid. All resistances were less than 6 K⍀. Electromyographic activity was amplified and digitized with a computerized EMG startle response monitoring system (SR-LAB; San Diego Instruments, San Diego, Caifornia). The EMG signal was filtered with low- and high-frequency cutoffs at 30 Hz and 1000 Hz, respectively. The system was set to record 250 1-msec readings starting at the onset of the startle stimulus. Subjects were seated and asked to look at the set of four lights mounted on the wall approximately 5 feet from their seat. All acoustic stimuli were delivered binaurally through headphones (model TDH-39-P; Maico, Minneapolis, Minnesota). The startle session (see diagram in Figure 1) began with a 1-min acclimation period consisting of 70-dBA broadband noise, which continued as the background noise throughout the session. The startle probe (noise burst) was either a 104- or 108-dBA sound pressure level, 40-msec burst of broadband noise with a near instantaneous rise time. According to methods established by Grillon and Davis (1997), the aversive stimulus (US) was a
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Figure 2. Diagram of the trials in the AX⫹/BX⫺ session. The AB and AC trials were structured the same as the BX⫺ trials. NA, noise alone.
100-msec airblast with an intensity of 140 psi directed to the larynx, emitted by a compressed air tank attached to polyethylene tubing and controlled by a solenoid valve. A, B, C, and X were green, purple, orange, or blue lights ranging in light transmission from 4.0% to 4.2% (counterbalanced color assignment across subjects). Figure 1 shows a diagram of the AX⫹/ BX⫺ startle session. The test session began with a habituation phase consisting of six startle probes (three at 104 dB and three at 108 dB) to reduce initial startle reactivity and rule out non-startlers. To minimize individual variability in baseline startle, subjects were either assigned to the 104-dB session or the 108-dB session, according to startle level in the habituation phase (if startle was below 100 machine units, subjects were assigned to 108 dB). The conditioning phase included six startle probes presented alone, six trials in which stimuli A and X were paired with the US (AX⫹), and six trials in which stimuli B and X were not paired with the US (BX⫺) (see Figure 1). The AX⫹ stimuli were presented serially within a trial, and the order of A and X alternated randomly across trials. In the AX⫹, the first light came on and stayed on for 7 sec; after 3 sec, the second light came on, so that for the last 4 sec the two lights were presented together. The startle probe was presented at the end of 6 sec (when the two lights had been presented together for 3 sec) and was followed by the airblast 500 msec later (see trial diagram in Figure 2). The airblast lasted for 250 msec, and the lights stayed on for another 250 msec after that, so that both lights were still on during both the startle probe and the airblast. In the BX⫺ trials, as well as in the AB and AC test trials, there was no airblast; therefore, the first light stayed on for 6 sec and 250 msec, and the second light was presented for the last 3250 msec. In these trials as well, the startle probe was presented at the end of the first 6 sec, and the lights stayed on for another 250 msec after the startle probe. The testing phase consisted of two blocks: each block included six startle probes presented alone and six presentations of AB or AC, respectively (see Figure 1). The order of the two blocks (i.e., one with AB trials and one with AC trials) was counterbalanced across subjects. In between the two blocks, there was a brief reconditioning phase in which AX⫹ was presented with the US again to maintain fear potentiation to AX. We did not repeat the presentations of BX because these trials were not reinforced and therefore habituation to the US was not a concern, whereas prolonging the testing session more than necessary would have raised the issue of startle habituation. In all phases of the experiment, intertrial intervals were of randomized duration, ranging from 9 to 22 sec. Response Keypad A response keypad unit (SuperLab; Cedrus, San Pedro, California) was incorporated into the startle session so that the EMG startle response monitoring system (SR-LAB) signaled the onset
of a light in the SuperLab software program. Each trial contained two light components (e.g., A and X). Subjects were instructed to respond to each light separately on each trial by pressing one of three buttons: one when they expected a light to be followed by the airblast, a second button when they did not expect the light to be followed by the airblast, and a third button when they were uncertain of what to expect. Awareness We assessed awareness of the experimental contingency according to the subjects’ keypad responses. First, the subjects had to have two consecutive correct responses to each of the two types of training trials (AX⫹ and BX⫺). The second criterion was to correctly label the last training trial (i.e., trial number 6) of each type. We operationally defined correct responses to AX⫹ trials as expectations of an airblast when presented with the A light as well as during an X light when it followed A; the correct responses to BX⫺ were expectations of no airblast on the B light as well as on an X light when it followed B. Subjects who did not meet both criteria were defined as unaware and excluded from further analyses. Statistical Analysis Repeated-measures analysis of variance (ANOVA) models were used to test effects of trial type (four levels: AX, BX, AB, and AC). The dependent variable was the difference score calculated by subtracting the startle amplitude during light presentation from the baseline startle amplitude, in this case the mean of the startle amplitudes on noise-alone trials (probes delivered in the absence of the light) during the conditioning and testing phase. Because of the variable nature of the startle response, we did not want to use a single data point for each individual and thus averaged three trials of each trial type. For AX⫹ and BX⫺ trial types we wanted to capture the results of training and therefore we calculated the mean of the last three trials of each type; for AB and AC trial types we wanted to test immediate transfer of safety (without the effects of learning) and thus used only the first three trials of each type. Furthermore, to account for habituation to the US throughout the session we calculated a mean AX, which included the re-conditioning block of AX, and subtracted the AB trial types and the AC trial types from the mean AX (AXAVG) to measure inhibition. Finally, to determine whether subjects transferred safety on the very first presentation of AB, we tested the linear effect of the mean AX and each of the first three trials of either AB or AC. To correct for violations of the sphericity assumption in the repeated-measures ANOVAs, we used Huynh-Feldt ε adjustments. Significant main effects of trial type were analyzed with specific contrast comparing AX with BX, AB with AX, AC with AX, and AB with AC. We also tested the four trial types for a www.sobp.org/journal
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T. Jovanovic et al was not [F (1,25) ⬍ 1, 2 ⫽ .02]. The data were also analyzed with order of testing (AB first vs. AC first) and decibel level (104 vs. 108) as between-groups variables. Neither the order of the testing blocks nor the decibel level interacted with the results. Conditioned Versus External Inhibition We looked at inhibition by subtracting the average startle amplitude on the first three AB or AC trial types from AXAVG (AX averaged between the second block of conditioning and the re-conditioning block to take into account habituation to the US, as described in Statistical Analysis). In this analysis, AB trials showed significantly more inhibition than the AC trials [F (1,25) ⫽ 4.79, p ⬍ .05, 2 ⫽ .16) (see Figure 4).
Figure 3. Startle potentiation to AX, BX, AB, and AC according to the difference score, calculated as the difference in startle to the acoustic probe in the presence and absence of the light cue.
quadratic trend. Effect sizes of the individual effects are reported with partial 2. All analyses were conducted with SPSS 10.0 for Macintosh (SPSS, Chicago, Illinois).
Results Of the 41 subjects who signed informed consent, 14 were excluded from further analyses because they were unaware of the experimental contingencies as measured by the response keypad (see Awareness section in Methods and Materials), and one was excluded owing to data collection error. This resulted in a final sample size of 26 subjects; 9 were female and 17 were male. Of the 26 subjects, 19 used the keypad as directed, pressing a button for each light; one female and six male subjects only pressed a button once per trial when the two lights were presented together. The testing blocks were evenly counterbalanced across subjects, with 13 receiving the AB testing block first, and 13 receiving the AC block first. Fear-Potentiated Startle Startle was robustly potentiated in the presence of all trial types, as indicated by the intercept term of the ANOVA; that is, the difference score between the trials with lights and the noise-alone trials was significantly different from zero [F (1,25) ⫽ 19.80, p ⬍ .001, 2 ⫽ .44]. Furthermore, there was an overall significant effect of trial type on the difference score [F (3,75) ⫽ 4.96, p ⬍ .01, 2 ⫽ .17]. We analyzed the specific trial type contrasts of AX versus BX, AX versus AB, AX versus AC, and AC versus AB. In the second block of conditioning we found significantly larger startle potentiation to the reinforced trials (AX) than to the non-reinforced trials (BX) [F (1,25) ⫽ 7.94, p ⬍ .01, 2 ⫽ .24] (see Figure 3). We also found significantly less potentiation to AB compared with AX [F (1,25) ⫽ 7.88, p ⬍ .01, 2 ⫽ .24], whereas there was no decreased potentiation to AC compared with AX. Comparing AB and AC directly, we found that AB was potentiated significantly less than AC [F (1,25) ⫽ 4.79, p ⬍ .05, 2 ⫽ .16] (Figure 3). We found that the subjects tended to habituate to the US throughout the session, so that startle potentiation to the second block of conditioning AX was slightly larger than to the reconditioning block of AX [F (1,25) ⫽ 3.29, p ⬍ .08, 2 ⫽ .12]. Thus, we decided to average these two values of AX (AXAVG) and test whether this more conservative analysis would also show AB inhibition. We again found that startle to AB was smaller than AXAVG [F (1,25) ⫽ 6.34, p ⬍ .05, 2 ⫽ .20], whereas startle to AC www.sobp.org/journal
Configural Versus Elemental Processing Transfer of inhibition of fear-potentiated startle to the AB trials suggests that the A and B lights were processed as elements rather than as configural stimuli; that is, A, rather than an A-and-X compound, was the excitatory stimulus, whereas B was the inhibitory stimulus. The data from the response pad indicate that this was the case: on the last training trial, A (in an AX trial) was labeled as reinforced by 89.5% of the subjects, whereas B (in a BX trial) was always labeled as nonreinforced [2(6) ⫽ 66.9, p ⬍ .001]. On the first testing trial, B (in an AB trial) was labeled as nonreinforced 94.7% of the time, whereas C (in an AC trial) was labeled as unknown 68.4% of the time. In addition, as further indication of transference, the first presentation of A in the testing trial (an AB trial, where B preceded A) was only labeled as reinforced 10.5% of the time, compared with 89.5% at the end of training [2(6) ⫽ 33.2, p ⬍ .001].
Discussion In this study, we sought to develop a new method of measuring inhibition of fear in humans that might reduce some of the disadvantages of the more commonly used methods, such as extinction and conditioned inhibition. To do this, we adapted a conditional discrimination procedure, of the form AX⫹/BX⫺, developed to measure fear inhibition in rats with the fearpotentiated startle paradigm (Myers and Davis 2004). Using lights of four different colors (A, B, C, X), we paired colors A and X with a blast of air to the throat, whereas colors B and X signaled that no shock would occur. To encourage subjects to use elemental rather than configural strategies during conditioning
Figure 4. Inhibition of potentiated startle to AB and AC according to a difference score from AXAVG; that is, subtracting the startle amplitude to AB (or AC) from the startle amplitude for AXAVG (the AX value was averaged between conditioning and reconditioning to account for habituation).
T. Jovanovic et al (Williams et al 1995), the subjects used a response keypad to label the reinforcement contingencies to each light in a trial as “safe,” “danger,” or “do not know,” so that A would be perceived as the danger signal and B would be perceived as the safety signal. The response keypad also allowed us to determine whether the subjects were learning the discrimination task on a cognitive level (i.e., whether they were aware of the contingencies in the experiment). We found that approximately 34% of the subjects were not aware of the contingencies in the experiment, according to their keypad performance, and excluded them from further analyses. Using potentiation of the eyeblink component of startle as our operational definition of fear, we found that the aware subjects startled more to AX than to noise alone and startled less to AB than to AX, indicating that they were both potentiating to the fear stimulus and inhibiting the fear response in the presence of safety signals. To demonstrate that B was indeed a safety cue, we had to show that subjects could immediately transfer safety on a subsequent AB test trial; that is, that the decrement in startle to AB relative to AX was not an effect of learning that AB was non-reinforced. We tested this by focusing on the first three presentations of AB and found that the subjects did indeed show immediate transfer on those trials. Furthermore, the response pad data indicate that the subjects were transferring safety on a cognitive level as well: on the very first presentation of AB, the subjects immediately recognized B as a safety signal and dramatically reduced the level of danger expected with A when it was paired with B. This represents the first study to demonstrate transfer in a conditioned inhibition experiment with fear-potentiated startle in humans. In the Grillon and Ameli (2001) study, in which a conditioned inhibition paradigm of the form A⫹, XA⫺ was used, no transfer of the safety cue (X) to a separately trained danger cue (B) was found, even when the investigators looked at as many as three blocks of B preceded by X. There are two possible reasons for the difference between Grillon’s study and our study. First, our experimental paradigm was designed to minimize second-order conditioning to the safety cue because B was never actually paired with A but instead was paired with X, which was shown in the rodent study to be much less fearful than A (Myers and Davis 2004); however, because Grillon found significant differences between the danger stimulus (A⫹) and the conditioned inhibitor (XA⫺), it is unlikely that X was second-order conditioned by A. The lack of transfer in the Grillon study could also be explained by configural processing, such that the AX compound trials were perceived by the subjects as a unique, new stimulus rather than as a combination of previously learned elements. Although Grillon used serial presentations of the stimuli to minimize configuration, that approach might not have been sufficient to induce elemental processing. In our study, startle to the combination of danger and safety (AB) was more inhibited than startle to the combination of danger and novelty (AC). We believe that the use of the response pad forced the subjects to perceive the stimuli as elements, which resulted in the transfer of safety and the subsequent inhibition of fear in our subjects. In addition, prior experience with compound stimuli seems to reduce external inhibition (Myers and Davis 2004), which might be another reason that we saw little external inhibition in the current study. Although we believe this AX⫹/BX⫺ design is superior to the typical A⫹, XA⫺ design, it still has the usual problems encountered with the use of fear conditioning in humans; namely, habituation to the aversive stimulus and a large proportion of
BIOL PSYCHIATRY 2005;57:1559 –1564 1563 unaware subjects (Grillon and Baas 2003). The problem of habituation can be remedied somewhat statistically; however, the unawareness issue requires testing larger sample sizes to replace subjects who do not learn the experimental contingencies. The AX⫹/BX⫺ paradigm allows for a more independent analysis of fear potentiation and fear inhibition and can be used in clinical populations to determine which of the two processes is dysregulated. For instance, PTSD patients might have normal levels of fear potentiation but have trouble inhibiting fear responses (Grillon and Morgan 1999). One of the advantages of the current study was the use of an airblast as the aversive stimulus, which is well tolerated by psychiatric patients and allows for larger group differences when comparing patients with control subjects (Grillon and Baas 2003). In future studies, this startle experiment can be used to test such patients, as well as to assess psychopharmacologic treatments to determine how they affect each process independently. In summary, we found that the AX⫹/BX⫺ paradigm, adapted from the animal model of conditional discrimination, can measure both fear potentiation and fear inhibition in human subjects under conditions in which the normal problems of second-order conditioning, configural learning, and external inhibition are minimized.
This research was supported by the Mental Health Service, Atlanta Department of Veterans Affairs Medical Center; the Science and Technology Center Program, the Center for Behavioral Neuroscience, of the National Science Foundation under Agreement No. IBN-9876754 (Venture grant, EJD); the American Psychiatric Association/Glaxo SmithKline (EJD); National Institute of Mental Health grants 1R24MH067314-01A1 (B. Rothbaum), R37 MH47840 (MD); Kirschstein National Research Service Award Individual Fellowship 1F32 MH070129-01A2 (TJ); and the Woodruff Foundation, Emory University School of Medicine. We gratefully acknowledge the input of Barbara Rothbaum, Kerry Ressler, Kim Huhman, Stephan Hamann, and Christian Grillon. Ameli R, Ip C, Grillon C (2001): Contextual fear-potentiated startle conditioning in humans: Replication and extension. Psychophysiology 38:383–390. Davis M (1992): The role of the amygdala in fear and anxiety. Ann Rev Neurosci 15:353–375. Davis M, Falls WA, Campeau S, Kim M (1993): Fear-potentiated startle: A neural and pharmacological analysis. Behav Brain Res 58:175–198. Falls WA, Davis M (1997): Inhibition of fear-potentiated startle can be detected after the offset of a feature trained in a serial feature negative discrimination. J Exp Psychol Anim Behav Process 23:3–14. First MB, Spitzer RL, Gibbon M, Williams JBW (1998): Structured Clinical Interview for DSM-IV Axis I Disorders-Patient Edition (SCID-I/P, Version 2.0, 8/98 revision). New York: New York State Psychiatric Institute. Grillon C (2002): Associative learning deficits increase symptoms of anxiety in humans. Biol Psychiatry 51:851– 858. Grillon C, Ameli R (2001): Conditioned inhibition of fear-potentiated startle and skin conductance in humans. Psychophysiology 38:807– 815. Grillon C, Baas J (2003): A review of the modulation of the startle reflex by affective states and its application in psychiatry. Clin Neurophysiol 114: 1557–1579. Grillon C, Davis M (1997): Fear-potentiated startle conditioning in humans: Effects of explicit and contextual cue conditioning following paired vs. unpaired training. Psychophysiology 34:451– 458. Grillon C, Morgan CA III (1999): Fear-potentiated startle conditioning to explicit and contextual cues in Gulf War veterans with posttraumatic stress disorder. J Abnorm Psychol 108:134 –142.
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