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- ARCHIVAL REPORTS Neurobiological Basis of Failure to Recall Extinction Memory in Posttraumatic Stress Disorder Mohammed R. Milad, Roger K. Pitman, Cameron B. Ellis, Andrea L. Gold, Lisa M. Shin, Natasha B. Lasko, Mohamed A. Zeidan, Kathryn Handwerger, Scott P. Orr, and Scott L. Rauch Background: A clinical characteristic of posttraumatic stress disorder (PTSD) is persistently elevated fear responses to stimuli associated with the traumatic event. The objective herein is to determine whether extinction of fear responses is impaired in PTSD and whether such impairment is related to dysfunctional activation of brain regions known to be involved in fear extinction, viz., amygdala, hippocampus, ventromedial prefrontal cortex (vmPFC), and dorsal anterior cingulate cortex (dACC). Methods: Sixteen individuals diagnosed with PTSD and 15 trauma-exposed non-PTSD control subjects underwent a 2-day fear conditioning and extinction protocol in a 3-T functional magnetic resonance imaging scanner. Conditioning and extinction training were conducted on day 1. Extinction recall (or extinction memory) test was conducted on day 2 (extinguished conditioned stimuli presented in the absence of shock). Skin conductance response (SCR) was scored throughout the experiment as an index of the conditioned response. Results: The SCR data revealed no significant differences between groups during acquisition and extinction of conditioned fear on day 1. On day 2, however, PTSD subjects showed impaired recall of extinction memory. Analysis of functional magnetic resonance imaging data showed greater amygdala activation in the PTSD group during day 1 extinction learning. During extinction recall, lesser activation in hippocampus and vmPFC and greater activation in dACC were observed in the PTSD group. The magnitude of extinction memory across all subjects was correlated with activation of hippocampus and vmPFC during extinction recall testing. Conclusions: These findings support the hypothesis that fear extinction is impaired in PTSD. They further suggest that dysfunctional activation in brain structures that mediate fear extinction learning, and especially its recall, underlie this impairment. Key Words: Amygdala, classical, conditioning, hippocampus, posttraumatic, magnetic resonance imaging, prefrontal cortex, stress disorders T he pathophysiology of posttraumatic stress disorder (PTSD) has been extensively studied over the past several years with neuroimaging and probes such as script-driven imagery and visual emotional stimuli (reviewed in [1– 4]). Studies have identified a network of dysfunctional brain regions, including amygdala; hippocampus; and subregions of the medial prefrontal cortex, including ventromedial prefrontal cortex (vmPFC) and dorsal anterior cingulate cortex (dACC). Individuals with PTSD typically show exaggerated amygdala and diminished hippocampal activation relative to control subjects (5–10). The dACC has emerged as another brain region that seems to be hyperactive in PTSD (11–13). Most studies have shown that vmPFC is hypoactive in this disorder (12,14 –21), but a few have reported hyperactivity (10,13,22–24). Although findings from these studies provide insight into the pathophysiology of PTSD, the function of these brain regions within the context of fear extinction learning and its recall (or retention) has not been directly examined. Extinction learning refers to the gradual, within-session decrements of conditioned fear responses, From the Department of Psychiatry (MRM, RKP, CBE, ALG, LMS, NBL, MAZ, SPO, SLR), Massachusetts General Hospital and Harvard Medical School, Boston; Department of Psychology (LMS, KH), Tufts University, Medford; McLean Hospital (SLR), Belmont, Massachusetts; and the Department of Veterans Affairs (SPO), Medical Center, Research Service, Manchester, New Hampshire. Address correspondence to Mohammed R. Milad, Ph.D., Department of Psychiatry, Harvard Medical School and Massachusetts General Hospital, 149 13th St., CNY 2614, Charlestown, MA 02129. E-mail: milad@nmr. mgh.harvard.edu. Received Mar 16, 2009; revised Jun 25, 2009; accepted Jun 26, 2009. 0006-3223/09/$36.00 doi:10.1016/j.biopsych.2009.06.026 whereas extinction recall refers to the retrieval and expression of the learned extinction memory after a delay (25). Understanding the basis of these processes is important, given that one of the main clinical characteristics of PTSD is exaggerated and persistent fear responses to reminders of the traumatic event. It is also important in that the current behavioral treatment of choice, exposure therapy, relies on extinction-based mechanisms (26,27). Pavlovian fear conditioning is commonly employed to probe the neurobiology of fear acquisition and its inhibition in rodents (28 –31), and it has also been used in psychophysiological (32–34) and neuroimaging studies of humans (35–37). In this procedure, conditioned responses (CRs) are formed when a conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (US), such as a mild electric shock. These CRs can then be diminished or extinguished by the repeated presentation of the CS in the absence of the US. Pavlovian fear conditioning and extinction are relevant to the neurobiology of PTSD, given that this disorder involves learned fear (27) that might persist for decades after the trauma exposure (38). Studying them might elucidate mechanisms by which perseverant fear responses occur. The hypothesis that extinction of conditioned fear is deficient in PTSD (3,39) is supported by de novo fear conditioning and extinction studies that have demonstrated deficient extinction learning (40). Moreover, we recently reported psychophysiological data indicating that Vietnam veterans diagnosed with PTSD have an acquired impairment in the retention of extinction memory (41). Neurobiological research has advanced our understanding of the mechanisms underlying extinction learning and recall. Numerous studies conducted in rodents with various pharmacological and molecular manipulations and electrophysiological and microstimulation tools have indicated that extinction learning and recall involve different cellular mechanisms and possibly different brain regions (for review, see [42]). For example, studies BIOL PSYCHIATRY 2009;66:1075–1082 © 2009 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved. 1076 BIOL PSYCHIATRY 2009;66:1075–1082 suggest that, in addition to its role in fear acquisition, the amygdala seems to be implicated in extinction learning, whereas the vmPFC (corresponding to the infralimbic cortex in rodents) and hippocampus seem to be involved in extinction recall (29,31,42– 44). In contrast, a region dorsal to the vmPFC in rats, viz., the prelimbic cortex, has been found to promote conditioned fear expression (45,46,47). Neuroimaging studies have recently examined extinction circuitry in healthy humans. In a study with functional magnetic resonance imaging (fMRI) (48), amygdala was activated during extinction learning, whereas vmPFC was activated during extinction recall. More recently, we reported that vmPFC and hippocampus are co-activated during extinction recall and that the degree of such activation is positively correlated with psychophysiologically measured extinction retention (49), as is vmPFC thickness (50). In contrast, thickness and functional activation of the dACC, homologous to rat prelimbic cortex, are correlated with expression of conditioned fear in humans (37). Thus, there is converging evidence in rodents and humans implicating the vmPFC and hippocampus in extinction recall and the dACC in fear expression. Finally, the amygdala seems to be involved both in fear expression and extinction learning, which might lead to ambiguous predictions. The objective of the present study was to examine the neurobiological basis of deficient extinction recall in PTSD with a focus on the aforementioned brain regions. While in a 3-T fMRI scanner, PTSD and trauma-exposed non-PTSD control (TENC) subjects underwent a 2-day Pavlovian fear conditioning and extinction procedure that we have previously used in healthy (39,51,52) and PTSD subjects (41). Skin conductance response (SCR), a commonly used measure in human fear studies (47,53) served as the dependent measure of conditioned responding. On day 1, subjects underwent fear conditioning to two pictures of differently colored lamps, followed by extinction for one of them. Day 2 tested recall of the extinction that had been learned the previous day by contrasting responses to the previously extinguished and unextinguished stimuli. Several hypotheses were tested. First, we predicted impaired extinction recall as measured by SCR in PTSD. Not only would this represent a replication of our previous report (41); it would also extend this finding to PTSD caused by civilian trauma. Second, we predicted lesser vmPFC activation during extinction learning in the PTSD group. However, no directional predictions were made for amygdala activation during extinction learning, because of its ambiguous role described in the preceding text. Third, we predicted lesser vmPFC and hippocampal activations and greater amygdala and dACC activations during (impaired) extinction recall in the PTSD group. Fourth, we predicted that the magnitude of extinction recall, indexed by percent extinction retention, would be positively correlated with vmPFC and hippocampal activations and inversely correlated with amygdala and dACC activations across all subjects. Methods and Materials Subjects A total of 19 PTSD patients and 20 trauma-exposed non-PTSD control subjects were recruited from the community. After a full explanation of the study’s procedures, written informed consent was obtained in accordance with the requirements of the Partners Healthcare System Human Research Committee. All subjects completed participation in the 2-day fear conditioning and extinction paradigm. Three PTSD and five TENC subjects were www.sobp.org/journal M.R. Milad et al. excluded from the data analysis because of excessive motion in the scanner. There remained 16 PTSD (6 women, 10 men) and 15 TENC subjects (8 women, 7 men, Fisher’s exact test p ! .48). Psychodiagnostics, Demographics, and Psychometrics The Clinician-Administered PTSD Scale (CAPS) conferred PTSD diagnostic status. The Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) determined the presence of other mental disorders. The TENC subjects with any current mental disorder were excluded. The PTSD subjects with current substance dependence were excluded, as were subjects who had used any psychotropic medication within 4 weeks before participation (1 year for neuroleptics). Type of trauma and current comorbid disorders appear in Table 1, as do group mean age, education, total CAPS scores, and age at first trauma exposure. Fear Conditioning, Extinction, and Testing Procedures The previously described (37,49) 2-day experimental protocol is summarized in Figure 1. All subjects selected a level of shock they regarded as highly annoying but not painful, to be used in the experiment. On day 1, during the Habituation phase, the to-be extinguished CS” (CS”E), unextinguished CS” (CS”U), and the CS that is never to be paired with the shock (CS#) (four of each) were presented in a counterbalanced manner within either the to-be conditioning or the to-be extinction context. During the Conditioning phase, two CS”s (e.g., red and blue lights) were depicted within a photograph of a distinct room (conditioning context), and each was paired with the US at a partial reinforcement rate of 60%. A third CS (e.g., a yellow light) was also depicted within the conditioning context but never paired with the US (CS#). There were 8 CS”Es, 8 CS”Us, and 16 CS# trials. When the shock US occurred, it followed the CS” offset without delay. The shock electrodes remained attached to the subject’s fingers during all subsequent phases, and subjects were instructed throughout the experiment (except during the Habituation phase) that they “may or may not receive the electric Table 1. Description of Demographics, Comorbidities, and Types of Trauma Exposure in Cohort Studied Demographics Age Education Mean age at trauma exposure CAPS Score Current Comorbidities (n) Major depression Panic disorder Alcohol abuse Other substance abuse Eating disorders Type of Trauma Exposure (n) Motor vehicle accidents Sexual assaults Physical assaults Childhood abuse Combat Witness to traumatic events PTSD TENC p 33.6 ($ 3.1) 15 ($ .53) 17 ($ 3.5) 66 ($ 6.04) 30.4 ($ 3.4) 15.9 ($ .74) 22.4 ($ 3.86) 10.5 ($ 2.66) .8 .43 .30 %.0001 5 2 1 2 2 0 0 0 0 0 2 8 4 6 3 3 2 2 6 1 0 4 The number of comorbid disorders and types of trauma shown might exceed the number of subjects, because a subject might have had more than one comorbid disorder or type of traumatic event. The $ symbol designates SEM. PTSD, posttraumatic stress disorder; TENC, trauma-exposed non-PTSD control subject; CAPS, Clinician-Administered PTSD Scale. BIOL PSYCHIATRY 2009;66:1075–1082 1077 M.R. Milad et al. A. Day 1 C di i i Conditioning Context B B. Shock Day 2 EExtinction i i LLearning i R Recallll 16 CS+E 8 CS+E 8 CS+U 16 CS- Context with CS 8 CS+E 8 CS+U 16 CS- 16 CS – Figure 1. Schematic of experimental protocol. (A) Pictures showing the visual contexts used in the experiment, within which conditioned stimuli (CS) were presented. In this example, pictures of an office and a conference room represent conditioning and extinction (E) contexts, respectively, whereas the blue light represents the CS” that was paired with the shock and later extinguished (CS”E). Extinction recall was conducted on day 2. (B) Schematic representation of the different phases of the experiment. A second CS” (red light, not shown) was presented during the conditioning phase but was not presented during the extinction learning phase (unextinguished, CS”U). The same CS” was then presented during the recall phase. A third light (yellow, not shown) was presented throughout the different phases of the experiment and was never followed with the shock (CS#). The numbers of each stimulus type presented during the conditioning, extinction learning, and extinction recall are indicated. Gray shading represents the extinction context. Habituation phase is not shown. shock.” However, shocks were only delivered during the Conditioning phase. After an approximate 1-min break, the Extinction Learning phase began. During this phase, the CS”E was depicted within a photograph of another distinct room (extinction context) and presented in the absence of the US, whereas the CS”U was not presented. There were 16 CS”E and 16 CS# trials. On day 2, during the Extinction Recall phase, 8 CS”E, 8 CS”U, and 16 CS# trials were again presented depicted within the extinction context. For each trial during the experiment, the context picture was presented for 9 sec: 3 sec alone followed by 6 sec in combination with the CS”E, CS”U, or CS#. The mean intertrial interval was 15 sec (range: 12 sec–18 sec). All experimental phases were conducted while blood-oxygen-level dependent (BOLD) signal data were being acquired via fMRI. Psychophysiological Measures As previously described (40,51,54), SCR for each CS trial was calculated by subtracting the mean skin conductance level during the 2 sec before CS onset (during which the context alone was being presented) from the highest skin conductance level during the 6-sec CS duration. Thus, SCRs to the CS”E, CS”U, and CS# reflected changes in skin conductance level beyond any change in SC level produced by the context. The magnitude of extinction retention (recall) was quantified as follows: each subject’s SCR to the first four CS” trials of the extinction Recall phase was divided by their largest SCR to a CS” trial during the Conditioning phase and then multiplied by 100, yielding a percentage of maximal conditioned responding. This in turn was subtracted from 100% to yield an “extinction retention index.” The purpose of calculating the extinction retention index was to normalize each subject’s SCR during extinction recall to that exhibited during the conditioning phase. This index is important, because it adjusts the SCR during extinction recall for differences in CR magnitude during acquisition. Unless otherwise specified, all data are presented as means $ SEM. Analysis of variance (ANOVA) and Student t tests were performed to test for statistically significant differences between means, with appropriate Bonferroni corrections when required. Image Acquisition The image acquisition parameters were identical to those previously used in our laboratory (49). Briefly, a Trio 3.0-Tesla whole body high-speed imaging device equipped for echo planer imaging (Siemens Medical Systems, Iselin, New Jersey) with an 8-channel gradient head coil was used. Head movement was restricted with foam cushions. After an automated scout image was obtained and shimming procedures were performed, high-resolution three-dimensional magnetization prepared rapid gradient echo sequences (repetition time/echo time [TR/TE]/flip angle ! 7.25 msec/3 msec/7°; 1 mm & 1 mm in plane & 1.3 mm) were collected for spatial normalization and positioning the subsequent scans. Scans with T1 (TR/TE/flip angle ! 8 sec/39 msec/90°) and T2 (TR/TE/flip angle ! 10 sec/48 msec/120°) sequences were used for registration of individual functional data. Functional MRI images (i.e., BOLD) were acquired with gradient echo T2*-weighted sequence (TR/ TE/flip angle ! 3 sec/30 msec/90°) (55). The T1, T2, and gradient-echo functional images were collected in the same plane (45 coronal oblique slices parallel to the anterior-posterior commissure line, tilted 30° anterior) with the same slice thickness (3 mm & 3 mm & 3 mm). The same scanning procedure was conducted on Day 2. Functional MRI Data Analysis Functional MRI data were analyzed with the Freesurfer Functional Analysis Stream (http://surfer.nmr.mgh.harvard.edu). All functional runs were motion-corrected with the Analysis of Functional Images motion correction tool, spatially smoothed (full-width-at-half-maximal ! 5 mm) with a three-dimensional Gaussian filter, and intensity-normalized to the low-level baseline. Images were manually inspected for motion artifact, and subjects with ‘2-mm total vector motion were excluded. Subjects’ functional runs were then individually registered to their anatomical volumes with FLIRT (Functional Magnetic Resonance Imaging of the Brain Linear Image Registration Tool, http:// www.fmrib.ox.ac.uk/fsl/flirt/index.html), and the registrations were visually inspected for accuracy. Estimates of the stimulus www.sobp.org/journal 1078 BIOL PSYCHIATRY 2009;66:1075–1082 Results Psychophysiological Responses During Fear Conditioning (Acquisition) An ANOVA revealed a significant Stimulus main effect (F ! 19.6, p % .001), with greater responses to the CS” (combined across the first four to-be CS”E and to-be CS”U trials) than to the CS# (combined across the first four trials) in the PTSD (.28 $ .07 (S vs. .07 $ .05 (S) and in TENC (.15 $ .04 (S vs. #.08 $ .05 (S) groups. Importantly, there were no group differences in conditioning, as evidenced by the absence of a significant Group main effect (F ! 2.8, p ! .10) or Group & Stimulus interaction (F ! .13, p ! .72). Functional MRI analysis was not conducted for this phase. Psychophysiological and fMRI Responses During Extinction Learning An ANOVA for the late extinction SCR data (last 12 CS”E vs. last 12 CS# trials) revealed no significant main effect of Stimulus (F ! 1.06, p ! .31) or Group (F ! 1.62, p ! .21) and no significant Group & Stimulus interaction (F ! 2.13, p ! .16), suggesting that comparable extinction learning had been achieved in both groups (Figure 2A). There was, regarding the fMRI data, a significant Group & Stimulus interaction in right amygdala, which was more reactive to the CS”E relative to the CS# in PTSD relative to TENC subjects (t ! 3.71, p ! .00025; Figure 2B). The Group & Stimulus interaction in vmPFC was www.sobp.org/journal A. 0.3 SCR (sqrt) 0.2 02 0.1 0.0 -0.1 01 B. CS+ CSPTSD CS+ CSTENC PTSD vs. TENC CS+ > CS- (late ex!nc!on learning) vmPFC C. % Sign nal Change effects at each voxel were made with an event-related design and by convolving the functional signal for each event with a canonical hemodynamic response function. The analysis included a linear correction to account for low-frequency drift. Statistical parametric maps were calculated according to a general linear model for the contrasts of interest across the time window (56). The contrast used for the Stimulus factor during the Extinction Learning phase was the last 12 CS”E versus the last 12 CS# trials in the Extinction Learning phase. Note that no US was delivered during this phase. The contrast used for the Stimulus factor during Extinction Recall phase was the first four CS”E versus the first four CS”U trials. These specific trials were selected for three reasons. First, their use minimizes the confound introduced by additional extinction learning that might take place during this phase and be especially reflected in responses to the latter trials. Second, electrophysiological data from rodents indicate that the vmPFC signals extinction recall only during the early portion of extinction recall (57). Third, we found that this contrast revealed the most robust activation of the vmPFC in our previous studies in healthy humans. Group & Stimulus interactions (i.e., PTSD vs. TENC contrasts on the Stimulus contrast maps) were analyzed separately for the Extinction Learning and Recall phases. Functional regions of interest (ROIs) were empirically defined as clusters of contiguous voxels exceeding the a priori statistical threshold in the following text. The BOLD signal values were extracted from these ROIs to calculate percent signal change. These values were then used for regression analyses with the extinction retention index. Coordinates for the peak voxels in each region were specified in terms of the Talairach atlas (58) to allow comparison with results of previous studies. We focused our fMRI data analysis a priori on the vmPFC, amygdala, hippocampus, and dACC, areas within which we employed a threshold of uncorrected, one-tailed p % .001. We used a more stringent threshold of p % .0001 for activations and deactivations in remaining brain regions. M.R. Milad et al. Amygdala 0.4 0.2 0.2 0.1 0.0 02 -0.2 PTSD TENC 0.1 0.0 -0.1 -0.4 -0.1 -0.6 -0.2 Figure 2. Responses during late extinction learning (last 12 of 16 trials). (A) Skin conductance responses (SCRs) to the conditioned stimulus (CS) that was previously paired with shock (CS”, dark shading) versus the CS that was never paired with shock (CS#, light shading) in posttraumatic stress disorder (PTSD) (red) versus trauma-exposed non-PTSD control (TENC) (black) subjects. (B) Group & Stimulus interaction in ventromedial prefrontal cortex (vmPFC) and amygdala, Talairach coordinates: x ! #15, y ! 34, z ! #21 for vmPFC and x ! 25, y ! #6, z ! #24 for amygdala. Image is masked to show only the activation in this hypothesized brain region. Threshold for displaying the images is set at p ! .01. (C) Percent signal change extracted from the amygdala and vmPFC functional region of interest shown in (B). marginally significant, showing deactivation to the CS”E relative to the CS# in PTSD relative to TENC subjects (t ! #3.28, p ! .0015; Figure 2B). Extracted percent BOLD signal changes from the amygdala and vmPFC functional ROIs are shown in Figure 2C. These data indicate that, during extinction learning, amygdala activation (to CS” relative to CS#) was observed in PTSD subjects, and amygdala deactivation was observed in TENC subjects. The opposite pattern was observed in the vmPFC, (i.e., deactivation in PTSD and activation in TENC). Psychophysiological and fMRI Responses During Extinction Recall An ANOVA for the early extinction recall SCR data (first four CS”E vs. first four CS”U trials) revealed a significant Group & Stimulus interaction (F ! 4.99, p ! .03). Whereas the TENC group exhibited smaller SCRs to the stimulus that had been extinguished during the previous extinction learning phase compared with the stimulus that had not been extinguished (.12 $ .07 (S for CS”E vs. .30 $ .1 (S for CS”U, F ! 5.14, p ! .03), the PTSD group did not (.40 $ .11 (S for CS”E vs. .37 $ .10 (S for CS”U, F ! 1.1, p ! .3), suggesting impaired recall of extinction memory in the PTSD group (Figure 3A). Consistent BIOL PSYCHIATRY 2009;66:1075–1082 1079 M.R. Milad et al. A. * SCR (sq qrt) 0.6 * 0.4 0.2 0.0 CS+E CS+U CS+E B. PTSD vs. TENC CS+E > CS+U L-vmPFC C. 0.6 % Signal Change CS+U 0.4 R-vmPFC Hippocampus Dorsal ACC 0.6 0.6 0.6 0.4 0.4 0.4 02 0.2 02 0.2 02 0.2 02 0.2 0.0 0.0 0.0 0.0 -0.2 -0.2 -0.2 -0.2 -0.4 -0.4 -0.4 -0.4 PTSD TENC Figure 3. Responses during early extinction recall (first four trials). (A) The SCRs to the stimulus that was previously extinguished on day 1 (CS”E, dark shading) versus the CS that was not extinguished on day 1 (CS”U, light shading) in PTSD (red) vs. TENC (black) subjects. (B) Group & Stimulus interactions. Talairach coordinates: left (L)-vmPFC: x ! #10, y ! 43, z ! #11; right (R)-vmPFC: x ! 2, y ! 45, z ! #12; hippocampus, x ! 32, y ! #9, z ! #27; dorsal anterior cingulate cortex (dACC): x ! #2, y ! 37, z ! 18. All images were masked to only show activations/deactivations in hypothesized brain regions. Threshold for displaying the images is set at p ! .01. (C) Percent signal change extracted from the functional regions of interest shown in (B). *p % .05. Abbreviations as in Figure 2. with this, the extinction retention index was significantly smaller in the PTSD than the TENC group (46% vs. 85%, t ! 2.9, p % .01). Moreover, within the PTSD group, total CAPS score was negatively correlated with extinction retention index (r ! #.71, p ! .01). With respect to the fMRI data during extinction recall, the same contrast was used (first four CS”E vs. first four CS”U trials). There were significant Group & Stimulus interactions in right hippocampus (t ! 4.27, p ! .0001), right vmPFC (t ! 3.54, p ! .0007), left vmPFC (t ! 3.41, p % .001), and left dACC (t ! 3.41, p % .001) (Figure 3B). Extracted percent BOLD signal changes from these functional ROIs are shown in Figure 3C. The TENC subjects showed activation in left and right vmPFC and hippocampus and deactivation in dACC, in response to the CS”E relative to the CS”U. The PTSD subjects showed the opposite patterns. To test for relationships between activations or deactivations in these brain regions during extinction recall and extinction memory, we conducted analyses correlating percent BOLD signal changes with extinction retention index across all subjects (Figure 4). These analyses revealed significant positive correlations between activation in vmPFC (bilaterally) and hippocampus and extinction retention as well as a trend toward a negative correlation between dACC activation and extinction retention. Activations/deactivations outside the a priori hypothesized brain regions are shown in Table 2. Subanalyses with Comorbidity-Free PTSD Subjects The key results were subjected to reanalysis excluding six PTSD subjects with current comorbid Axis I disorders. This analysis revealed that the Group & Stimulus interaction remained significant for the SCR data during extinction recall (F ! 5.39, p ! .02). Moreover, the percent extinction retention between the two groups remained statistically significant (52% for PTSD vs. 85% for TENC, t ! 2.18, p ! .037). Regarding the fMRI data, reanalysis of the main contrast during extinction recall (CS”E vs. CS”U) revealed that the deactivation in the bilateral vmPFC in the PTSD relative to the TENC group was marginally significant (t ! 3.25, p ! .0015 for both right and left vmPFC), whereas the hippocampal difference between groups remained significant (t ! 4.05, p ! .00025). The increased activation in the dACC in the PTSD relative to the TENC group became more significant (t ! 4.20, p ! .00015). The reduced significance level regarding the vmPFC activation is most likely due to reduced power. Thus, this subanalysis revealed that comorbidity in the PTSD sample analyzed in this study is unlikely to have accounted for the differences observed between groups with regard to either the psychophysiological or the fMRI data. Discussion The psychophysiological and fMRI data obtained in the TENC group show intact fear extinction memory (or recall), manifest in lower SCRs to a previously extinguished compared with a previously unextinguished CS that is associated with vmPFC and hippocampal activation during extinction recall, thereby replicating our previous report (49). In contrast, the psychophysiological data obtained in the PTSD group show impaired extinction www.sobp.org/journal 1080 BIOL PSYCHIATRY 2009;66:1075–1082 M.R. Milad et al. L-vmPFC Hippocampus % Exxt. Reten!on 100 50 0 -50 -1.0 -0.5 0.0 0.5 1.0 r = 0.45* P = 0.01 150 % Exxt. Reten!on r = 0.49* p = 0.005 150 100 50 0 -50 -1.0 % Signal Change % Ext. Reten!on 100 50 0 0.0 0.5 0.0 0.5 1.0 1.0 D orsal ACC 150 % Ext. Reten!on r = 0.45* 01 P=0 0.01 150 -0.5 -0.5 % Signal Change R-vmPFC -50 -1.0 PTSD TENC % Signal Change r = -0.33 0 055 p = 0.055 100 50 0 -50 -1.0 -0.5 0.0 0.5 1.0 % Signal Change Figure 4. Regression plots between percent extinction (Ext.) retention and percent blood-oxygen-level dependent signal change during extinction recall extracted from the functional regions of interest shown in Figure 2, collapsed across groups. All p values listed in the figures are below the Bonferroni correction threshold. Abbreviations as in Figures 2 and 3. retention, manifest in no difference between SCRs to the extinguished and unextinguished CSs, replicating another of our previous reports (41). In addition, the present data suggest that this deficient extinction retention in PTSD might be the result of dysfunctional responding in brain regions previously reported to be implicated in the recall of fear extinction in healthy subjects. Specifically, we found less activation in hippocampus and bilateral vmPFC but more activation in dACC during extinction recall in PTSD compared with TENC subjects. The amount of extinction retention across all subjects was positively correlated with activation in both vmPFC and hippocampus and nearly significantly negatively correlated with activation in dACC, thereby replicating prior fMRI results in an independent sample of healthy subjects and extending them to PTSD (48,49,59). The greater activation in the amygdala in PTSD patients during extinction learning replicates a recent report (16). However, despite their greater amygdala activation and their lesser vmPFC activation, the PTSD group displayed extinction learning that was comparable to the TENC group. Normal extinction learning in the PTSD group in the absence of vmPFC activation is consistent with animal studies. For example, it has been previously shown that lesions or pharmacological manipulations of the vmPFC do not interfere with extinction learning per se (28). Rather, single neurons recorded from this brain region increase their neural activity to the extinguished CS” only during extinction recall (57). Thus, the data gathered from the current study provide a translational link between rodent and human data indicating that vmPFC function is not necessary for initial extinction learning but is critical for extinction recall. In other words, the present data suggest that dysfunctional brain activation in the PTSD group (i.e., greater activity in amygdala, and lesser activity in vmPFC compared with the TENC group) during extinction learning might contribute to PTSD patients’ failure to consolidate extinction memory. The present data further suggest that failure to activate vmPFC and hippocampus during recall contribute to deficient expression of extinction memory in PTSD. As noted in the introduction, PTSD patients’ failure to activate these brain regions has also been found in other neuroimaging tasks. Table 2. Significant Activations in Regions Outside the A Priori Regions of Interest Area of Activation Late Extinction Learning Regions outside the a priori areas Superior temporal cortex (R) Superior temporal cortex (R) Superior temporal cortex (L) Extinction Recall Regions outside the a priori areas Cerebellar cortex (R) Cerebellar cortex (R Cerebellar cortex (R) Medial parietal cortex (R) Occipital cortex (L) Talairach Coordinates p Contrast: PTSD ‘ TENC, Late CS”E vs. CS# 61, #11, #4 64, #9, 0 #51, 5, #8 Contrast: PTSD ‘ TENC, Early CS”E vs. CS”U 3.1 & 10#5 4.3 & 10#5 7.9 & 10#5 22, #46, #14 9, #59, #40 40, #67, #34 #4, #17, 61 #48, #69, #1 4.0 & 10#6 1.2 & 10#5 2.8 & 10#5 3.0 & 10#5 4.7 & 10#5 Threshold for peak voxel p % 10#4, two-tailed, uncorrected. CS, conditioned stimulus; E, extinction; PTSD, posttraumatic stress disorder; TENC, trauma-exposed non-PTSD control subject; U, unextinguished. www.sobp.org/journal M.R. Milad et al. The dACC has traditionally been implicated in conflict monitoring, attention, and pain (60 – 62). One caveat when comparing the results of those studies and the data presented in the present study is that the term “dACC” has been used to refer to a broad area of the anterior cingulate. In a recent meta-analysis, Vogt et al. (60,61) identified a subregion of the dACC (termed the anterior midcingulate [aMCC]) that was specifically activated by fear-inducing stimuli. Importantly, the dACC region that showed activation during extinction recall in our PTSD subjects seems to overlap with aMCC. Moreover, we have previously shown that dACC thickness and function are positively correlated with conditioned responding during fear acquisition in healthy control subjects, suggesting that this brain region might be involved in promoting the fear response (37). A recent neuroimaging study reported increased activation of the dACC region in PTSD patients (11). All of these findings support a role for the dACC in the pathological expression of conditioned fear in PTSD. We observed, in addition to the a priori ROIs, increased cerebellar activation in PTSD patients relative to control subjects during extinction recall (Table 1). The meaning of this finding is unclear, given that we previously observed cerebellar activation during extinction recall in a healthy cohort (49). In addition to the well-documented role of this brain region in movement and motor coordination, the cerebellum has been reported to be involved in the processing of fear memories (63,64) and in extinction of eye-blink conditioning (65). Further studies are needed to clarify its role in emotional learning and memory, including fear extinction, in general, and in PTSD. It has been hypothesized that fear extinction and its retention are deficient in PTSD due to failure to activate brain extinction circuitry, including hippocampus and vmPFC (31,42). With positron emission tomography, Bremner et al. (16) were the first to examine fear conditioning and extinction learning in PTSD. The authors reported increased amygdala and decreased vmPFC activity in PTSD relative to control subjects, which is consistent with this hypothesis. In the current study, the link here between deficient, psychophysiologically measured extinction recall in PTSD and failure to activate vmPFC and hippocampus during extinction recall provide direct data in support of this model. The present results also provide neurobiological evidence that the pathologically elevated and persistent conditioned fear clinically observed in PTSD is at least in part due to failure to activate vmPFC and hippocampus as well as to hyperactivation of dACC and amygdala. The work was supported in all aspects, including design, data collection, and preparation, by a grant from the National Institute of Mental Health (1R21MH072156-1) to SLR and a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression to MRM. We would like to thank Dr. Clas Linnman for helpful comments on the manuscript. Scott Rauch’s financial disclosures are as follows: he received funded research through Massachusetts General Hospital (MGH) for Brain Stimulation Therapy from Medtronics; funded research through MGH for vegal nerve stimulation from Cyberonics; and funded research through MGH on anxiolytic action from Cephalon. 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