Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

The overall goal of the following experiment is to examine the neural substrates of aware and unaware fear learning and memory by simultaneously measuring skin conductance response, conscious unconditioned stimulus expectancy and functional MRI. This is achieved by measuring skin conductance response or SCR as an index of fear learning and memory. Unconditioned stimulus expectancy is also monitored, which reflects the participant's expectation of receiving the aversive uncondition to stimulus and can be used as an indicator of the participant's awareness of stimulus contingencies.

Further, FMRI data are acquired in order to identify learning related differences in brain activity. When subjects are aware compared to unaware of the relationship between stimuli, results typically show greater hippocampal responses to the positive conditioning stimulus or CS plus than to the negative conditioning stimulus or CS minus on perceived but not unperceived trials. However, amygdala responses are larger to the CS plus than CS minus on both perceived and unperceived trials.

The main advantage of this technique over other methods of assessing awareness such as post experimental questionnaires, is that awareness can be assessed on a trial by trial basis during the conditioning session. This method can help us answer key questions related to learning memory and emotion, such as what role contingency awareness plays in fear conditioning. Physiological monitoring systems are non-standard equipment in most imaging facilities.

Therefore, schedule 15 to 30 minutes prior to participant arrival to set up physiological monitoring and all other equipment described in this protocol. First, connect to control room computer operating. Acknowledge physiological monitoring software to the BioPack MP one 50 system using a standard ethernet crossover cable.

Next, connect the BioPack GSR amplifier to the RF interference Filter within the control room using a shield at extension cable. Also connect the RF interference filter to a shielded extension cable within the MRI scanning chamber. Connect the shielded extension cable to carbon fiber lead wires that will attach to the radio translucent electrodes.

Note that twisting the leads in a tight spiral can reduce artifacts in the skin conductance data that can be created during scanning. Now set up the radio translucent electrodes that will be attached to the distal phalanx of the middle and ring fingers of the participant's left hand while running the FMRI experiment. Next, the behavioral response and presentation software and hardware will need to be set up first.

Use a mini USB cable to connect the control room computer operating presentation software to the joysticks fiber optic response pad interface unit. Then connect a fiber optic cable to the fiber optic interface unit within the control room and pass the cable through a wave guide into the MRI chamber. In the scanner room, connect the fiber optic cable to the RI compatible joystick.

Next, connect the control room computer operating presentation software to the external VGA and audio ports of the control room console. Be sure to check the fiber optic cable connections between the FMRI control room console and the FMRI peripheral interface unit within the MRI chamber, as well as the connections between the peripheral interface unit and the audio visual display unit. Now place the audio visual display unit behind the head coil such that the participant will be able to view the monitor through an attached mirror.

Then use vinyl tubing to connect the display unit's acoustic interface box to the MR compatible stereo headphones. Finally, calibrate the volume of auditory stimuli using a sound pressure level meter. When the participant arrives, explain the details of the FMRI experiment and scanning procedure.

Then have him or her fill out a screening form for MRI safety and sign a research consent form. Inform the participant that two tones will be presented several times during the study and that the volume of the tones will vary above and below their perceptual threshold. These tones are the CS or conditioned stimuli.

I direct the participant to push a button on the joystick box immediately upon hearing either tone, and then indicate their expectation that an unconditioned stimulus or UCS will be presented. Next, by moving the joystick to control the position of a rating bar on a zero to 100 scale, inform the participant that ratings of zero indicate that he or she is certain that the unconditioned stimulus will not be presented. While ratings of 50 indicate uncertainty whether the UCS will be presented and ratings of 100 indicate certainty, the UCS will be presented.

Other values on the scale indicate intermediate expectations. Next, set the participant up in the scanner. Place electrodes on the distal phalanx of the middle and ring fingers of the participant's left hand and direct him or her to place the joystick in a comfortable and easy to reach position.

Due to the nature of scanning equipment MRI chamber room temperatures are often below 21 degrees Celsius. So cover the participant with a blanket to maintain temperature. Once the subject is positioned in the scanner, set imaging parameters to collect bold FMRI of the whole brain during the conditioning procedure.

36 4 millimeter thick slices should be sufficient to cover the brain with relatively standard imaging parameters. Now expose the participant to a differential fear conditioning procedure using two tones at 700 and 1300 hertz 10 seconds duration, 20 seconds ITI as the conditioned stimuli and allowed white noise, 100 decibels, 500 milliseconds as the unconditioned stimulus present 60 trials of CS plus co terminating with the unconditioned stimulus and 60 trials of CS minus presented without the unconditioned stimulus in a pseudorandom order such that no more than two trials of the same CS are presented consecutively counterbalance the tones that serve as the two stimulus types across participants. The presentation software should be programmed to modulate the volume of the CS plus and CS minus independently, such that the CS volume is adjusted on the subsequent trial with the same stimulus volume should be decreased five decibels if a button presses made meaning the trial was perceived and increased.

Five decibels. If a button press is not made following an unperceived trial sample skin conductance during the experiment at 2000 hertz using the acknowledged software and the MR compatible BioPack physiological monitoring system previously described. Also, sample and record unconditioned response expectancy data using presentation software.

Once all data is collected, obtain a standard high resolution T one weighted structural image such as an MP rage to serve as an anatomical reference for overlay of functional data. To begin analysis of the skin conductance data, first to apply a one hertz infinite impulse response or IIR low pass digital filter to the data to reduce artifacts produced during imaging. Then re-sample the skin conductance data at 250 hertz.

Next, calculate skin conductance response as the difference in skin conductance level from response onset to response peak. This data can be square root transformed to normalize the distribution of response amplitudes prior to statistical analysis. To determine the unconditioned stimulus expectancy, calculate the average of a one second sample response during the last second of conditioned stimulus presentation.

To begin FMRI analysis. First, complete standard pre-processing of brain imaging data, including slice, timing correction, image registration, and spatial smoothing using a functional imaging analysis software package. Next, create standard nuisance and stimulus based regressors for perceived and unperceived trials of the CS plus and CS minus, as well as the uncondition stimulus.

Create a motor response based reference waveform to serve as a nuisance regressor to account for motor activity related to button press responses. To do this, create a stick function that codes for the timing of button press responses. Then con, involve this function with a canonical hemodynamic response function.

Also create a motor response based reference waveform to serve as a nuisance regressor to account for motor activity related to joystick responses. Create a stick function that codes for timing of changes in the slope and involve the joystick slopes stick function with a canonical hemodynamic response function. Next, perform first level analyses using all stimulus based and nuisance regressors.

Then perform a second level repeated measures a NOVA to identify regions in which act. Activation shows a main effect of CS type, a main effect of perception or a CS type by perception interaction. The methodology presented here.

Typically a results in relatively high UCS expectancy ratings during perceived CS plus trials and low ratings during perceived CS minus trials. Such results indicate participants are aware of CS UCS contingencies on unperceived trials. UCS expectancy ratings typically remain unchanged from pre CS ratings.

In contrast, learning related changes in SCR are typically observed during both perceived and unperceived conditioning trials. Specifically, SCRs are larger to the perceived CS plus than to the perceived CS minus. Similarly larger SCRs are observed during unperceived CS plus than unperceived CS minus trials.

The functional imaging research using this methodology has demonstrated learning related hippocampal activation on perceived but not unperceived conditioning trials. In contrast, differential amygdala activity has been observed on both perceived and unperceived conditioning trials. Following this procedure, additional analyses can be performed in order to answer questions such as the effect that UCS expectancies have on the brain's response to aversive unconditioned stimuli.

After watching this video, you should have a good understanding of how to investigate aware and unaware fear memory by simultaneously measuring skin conductance response conscious UCS expectancies and functional MRI signal responses.