Department of Psychology, University of Alabama at Birmingham
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Knight, D. C., Wood, K. H. Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI. J. Vis. Exp. (56), e3083, doi:10.3791/3083 (2011).
Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior.
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.
The Biopac Systems, Inc. physiological monitoring system (see Table of specific equipment) is non-standard equipment in most imaging facilities. Schedule 15-30 minutes prior to participant arrival to set up physiological monitoring and other equipment described in this protocol (Figure 1).
2. Behavioral Responses (Joystick)
3. Stimulus Presentation
4. Experimental Procedure
5. Scanning Procedure
6. SCR Data Acquisition & Analysis
7. UCS Expectancy Data Acquisition & Analysis
8. Functional MRI Data Acquisition & Analysis
9. Representative Results:
The methodology presented here typically results in relatively high UCS expectancy ratings during perceived CS+ trials and low ratings during perceived CS- trials (Figure 5) 10,15,19. Such results indicate participants are aware of CS-UCS contingencies. On unperceived trials, UCS expectancy ratings typically remain unchanged from pre-CS ratings. UCS expectancies on these unperceived CS+ and CS- trials typically fall near 50 indicating participants are unsure of whether the UCS will be presented 10,15,19 (Figure 5). This inability to produce differential UCS expectancy ratings to the unperceived CS+ and CS- indicates that participants are unable to express their contingency awareness on unperceived conditioning trials (Figure 6). In contrast, learning-related changes in SCR have been observed during both perceived and unperceived conditioning trials 10,15,19. Specifically, SCRs were larger to the perceived CS+ than to the perceived CS-. Similarly, larger SCRs have been demonstrated during unperceived CS+ than unperceived CS- trials 10,15,19 (Figure 6). Taken together, these behavioral and autonomic data demonstrate fear conditioning with contingency awareness on perceived trials, and fear conditioning without contingency awareness on unperceived trials. The functional imaging research using this methodology has demonstrated learning-related hippocampal activation on perceived, but not unperceived conditioning trials15 (Figure 7). In contrast, differential amygdala activity was observed on both perceived and unperceived conditioning trials 15. These findings are consistent with the view that the hippocampus supports processes related to contingency awareness, while the amygdala supports CR expression with and without awareness.
Figure 1. Diagram of basic equipment for stimulus presentation and behavioral/psychophysiological response monitoring. Presentation software is used to present audio-visual stimuli and monitor UCS expectancy ratings made by moving a joystick with the right hand. AcqKnowledge software and Biopac equipment are used to monitor skin conductance from the left hand. Solid (Biopac), single dashed (IFIS Audio-Visual), and double dashed lines (Fiber optic joystick) depict cables for distinct stimulus presentation and response monitoring systems. Black arrows indicate direction of information flow.
Figure 2. Conditioned Stimuli. Present the CS+ and CS- in a pseudorandom order such that no more than 2 trials of the same CS are presented consecutively. Vary the volume of the CS+ and CS- independently. If a CS is perceived (indicated by a button press), decrease CS volume 5dB on the subsequent trial of the same CS. If a CS is unperceived (indicated by no button press), raise CS volume 5db on the subsequent trial with the same CS.
Figure 3. UCS expectancy rating scale. Instruct participants to rate their expectation of UCS presentation on a 0 to 100 scale. Ratings of 0 indicate certainty the UCS will not be presented, ratings of 100 indicate certainty the UCS will be presented, and ratings of 50 reflect uncertainty as to whether the UCS will be presented. Intermediate ratings should be used to indicate gradations in UCS expectancy.
Figure 4. Comparison of raw and filtered skin conductance data. a) Raw skin conductance data collected during fMRI. b) Skin conductance data after application of a 1Hz IIR low pass filter.
Figure 5. UCS expectancy ratings. -Participants typically report high UCS expectancies on perceived CS+ trials and low expectancies on perceived CS- trials. UCS expectancies on unperceived CS+ and CS- trials do not differ.
Figure 6. UCS expectancy and SCR. Differences in UCS expectancy are typically observed on perceived CS+ and CS- trials indicating participants are aware of the stimulus contingencies. On unperceived trials, UCS expectancy ratings typically do not differ indicating participants are unable to express their contingency awareness. In contrast, differences in conditioned SCRs are usually observed on both perceived and unperceived conditioning trials. Such findings reflect learned fear expression with (i.e. on perceived trials) and without (i.e. on unperceived trials) contingency awareness.
Figure 7. Functional MRI of the hippocampus and amygdala. Hippocampal responses are typically larger to the CS+ than CS- on perceived, but not unperceived conditioning trials. Differential amygdala responses are typically observed on both perceived and unperceived conditioning trials. These findings are consistent with the view that the hippocampus supports processes related to contingency awareness, while the amygdala supports fear expression with and without awareness.
The fear conditioning methodology described here provides a means to investigate the neural mechanisms of aware and unaware fear memory processes. This method takes advantage of the simultaneous monitoring of behavioral, autonomic, and fMRI data. Monitoring behavioral (i.e. UCS expectancy) and autonomic responses (i.e. SCR) is a critical component of this method. UCS expectancy provides a means to assess contingency awareness, while SCR provides an index of CR expression. Together, these behavioral and autonomic responses can be used during the presentation of supra and subthreshold CS+ and CS- trials to investigate fear conditioning with and without contingency awareness. Functional MRI data can then be used to investigate the neural correlates of aware and unaware fear memory processes. A particular strength of this methodology is that it exposes participants to each type of conditioning trial (i.e. perceived CS+ & CS-, unperceived CS+ & CS-). Within-subject designs like the one described here are more powerful than between subject designs because of the relatively large inter-subject variability observed in both SCR and fMRI signal responses. Another strong point of this method is that the volume of CS presentation is tailored to each participant's perceptual threshold. Further, the perceptual threshold is allowed to vary over the course of the conditioning session. Prior work has typically presented stimuli at a set level below threshold 7,20,21. However, perceptual thresholds can vary over time reducing the ability to detect subthreshold effects 22. An additional strength of this methodology is that UCS expectancy is assessed on a trial-by-trial basis during the conditioning session. Other fMRI research has assessed awareness of CS-UCS contingencies during post-conditioning evaluations 23. However, post-conditioning assessments 1) cannot assess variations in expectancy from trial-to-trial, 2) may be insensitive to subtle evidence of contingency awareness, and 3) are susceptible to issues that distort results such as forgetting and interference. Although there are a number of strengths to our methodology, monitoring UCS expectancy as described may engage attentional processes in a manner that differs from studies that do not use online expectancy measures. This is an issue that investigators should consider along with the advantages of this methodology when designing their projects.
No conflicts of interest declared.
Support provided by the University of Alabama at Birmingham Faculty Development Grant Program.