This research was supported by Grants R01AA15384 from the National Institute on Alcohol Abuse and Alcoholism and 5R01DA033809-02 from the National Institute of Drug Abuse to John J. Curtin.

The local ethics committee has approved the following procedure and all participants who have taken part in this procedure have given informed consent. For additional detail of psychophysiological measurement and stimulus presentation please see30,27.
1. Electromyography (EMG) Recording Preparation
<ol>
	<li>Ask the participant to wash their face thoroughly with soap, paying particular attention to the target sensor locations, which are located below one eye and in the middle of the participant&#8217;s forehead (see Figure 2).</li>
	<li>Seat the participant in a comfortable upright chair in the experimental chamber.</li>
	<li>Prepare the participant&#8217;s skin for EMG measurement.
	<ol>
		<li>Clean the target sensor locations with an alcohol pad.</li>
		<li>Clean the same locations with a gritty exfoliate gel using a small gauze pad to further remove dirt or dead skin cells that can impede measurement of the electromyographic activity.</li>
	</ol>
	</li>
	<li>Prepare and attach EMG electrodes.
	<ol>
		<li>Fill all silver-silver chloride (Ag&#8211;AgCl) sensor cups with conductive gel using a syringe and blunt needle.</li>
		<li>Attach a large (e.g., 8 mm) Ag-AgCl sensor to the center of the participant&#8217;s forehead using an adhesive collar.</li>
		<li>Attach two additional small (e.g., 4 mm) Ag-AgCl sensors below the participant&#8217;s eye using adhesive collars. Place the first of these small sensors in line with the pupil at forward gaze and the second sensor 1-2 cm lateral to the first (Figure 2; see also27). Do not allow the adhesive collars to overlap as this may increase movement artifact. Prevent gel overflow to avoid forming a gel bridge between the two sensors below the eye as this will cause current to flow via the bridge and impair measurement of EMG activity.</li>
	</ol>
	</li>
	<li>Start the EMG acquisition software on the physiology computer and ask the participant to blink a few times to verify that the EMG response is being recorded properly and that eye blinks can be observed on the display of the data collection software (see Figure 3A for an example of EMG activity associated with a blink).</li>
	<li>Check the impedance for each sensor. 
	NOTE: Many laboratories require impedances below 10 k&#937; (or more conservatively, 5 k&#937;) but actual tolerable thresholds for measured impedance levels depend on many variables such as experimental design, amplifier design, and practical constraints with respect to time needed to reduce impedances and the participant population. Regardless, high impedances increase susceptibility of the EMG signal to electrical artifact, which may be problematic (60 Hz noise; see Figure 3B).</li>
	<li>Place the headphones on the participant&#8217;s head.</li>
</ol>
2. Baseline Measurement of General Startle Reactivity
NOTE: This assessment also serves to further habituate the startle response prior to the three habituation probes delivered just before task start31. Including general startle reactivity as a covariate in statistical analysis of startle potentiation increases statistical power to detect within and between participant effects. General startle reactivity may also reflect an interesting individual difference measure12,32.
<ol>
	<li>Ask the participant to get comfortable before the start of the baseline task and to remain as still as possible throughout the task with their feet flat on the floor. Participant movement may introduce artifact into the EMG signal (see Figure 3C).</li>
	<li>Remind the participant that they can discontinue their participation at any point during the experiment. Monitor the participant using video and audio feed from the experimental chamber during both the baseline assessment and main task.</li>
	<li>Save the EMG signal with acquisition software on the physiology computer and start the stimulus presentation software on the stimulus control computer.</li>
	<li>Present the participant with a series of colored squares that will be used in the main task but have not yet been paired with electric shock. Present startle probes during a subset of these cues and the interval between the cues. Timing parameters for cue duration, interval between cues, and startle probes should match parameters from the main task. Reliable measurement of general startle reactivity requires the presentation of at least 4 probes. This baseline task takes approximately 5 min to complete.</li>
	<li>Average together the participant&#8217;s peak EMG startle response to each startle probe in the baseline procedure to produce one value which will serve as this participant&#8217;s general startle reactivity (see steps 6.1-6.6 for how to process the EMG data). Include general startle reactivity as an additive or interactive covariate in statistical models involving startle potentiation (see step 6.8).</li>
</ol>
3. Shock Tolerance Threshold Assessment
<ol>
	<li>Affix two shock electrodes with standard medical tape to the participant&#8217;s hand (e.g., distal phalanges of index and ring fingers of the hand)33&#8211;35.</li>
	<li>Present the participant with a series of increasingly intense electric shocks. After each shock is administered, ask the participant to rate how aversive they found the shock on a 100-point scale. Ask them to use a rating of 0 if they cannot feel a shock at all, a rating of 50 for the first level of shock that they consider to be uncomfortable, and a rating of 100 for the highest level of shock that they can tolerate.</li>
	<li>Instruct the participant that it is important to accurately report the highest shock they can tolerate. The participant should not be informed that their report will impact the actual shocks they receive as this may lead to bias in their report.</li>
	<li>Stop the shock tolerance assessment once the participant rates a shock as 100. Record the shock level and administer shocks at this level in the Threat Probability Task to control for individual differences in shock sensitivity. 
	NOTE: Electric shocks are administered to each participant&#8217;s subjective maximum shock tolerance threshold. However, lower intensity shocks are also used21. Regardless, it is important that the shock intensity selected is sufficient to elicit a robust negative affective response and associated startle potentiation from all participants.</li>
</ol>
4. The Threat Probability Task
<ol>
	<li>Provide the participant with a cover story that encourages attention throughout the task. 
	NOTE: Some participants may find it difficult to maintain attention throughout the Threat Probability Task. An example of a cover story that researchers can tell participants in order to encourage attention in this task is to tell the participant that the researchers are interested in measuring the participant&#8217;s ability to pay attention over time during a simple, repetitive visual task similar to the task required of air traffic controllers.</li>
	<li>Provide the participant with general task information and specific cue-shock contingencies for each condition.
	<ol>
		<li>Instruct the participant that the task lasts approximately 20 min.</li>
		<li>Instruct the participant that the task includes cues that last 5 sec each separated by 15-20 sec on average.</li>
		<li>Inform the participant that the cues are organized into sets with each set lasting 2-3 min each.</li>
		<li>Instruct the participant that there are three types of sets, 20% shock sets, 100% shock sets and No shock sets.</li>
		<li>Instruct the participant that they will receive shocks at the end of approximately 1 out of every 5 cues in 20% shock sets and 5 out of every 5 cues in 100% shock sets.</li>
		<li>Assure the participant that they will receive no shocks at any time during No shock sets or during the time between the presentations of the cues (ITI) in any of the sets.</li>
		<li>Allow the participant to ask questions about the task at the end of the instructions. Following this, quiz the participant to make sure they completely understand the shock contingencies. Remind the participant that they can discontinue their participation at any point during the experiment.</li>
	</ol>
	</li>
	<li>Save the EMG signal with acquisition software on the physiology computer and start stimulus presentation software on the stimulus control computer that will control task stimuli.</li>
	<li>Carefully monitor the participant for voluntary movements, closing of eyes, or excessive discomfort.</li>
</ol>
5. Post-experiment
<ol>
	<li>After the cued threat task, administer a questionnaire to the participant to verify that the threat contingencies were well understood during the task. Ask the participant to rate how anxious or fearful they were when they saw each threat cue on a 5 point rating scale from 1 (not at all anxious/fearful) to 5 (extremely anxious/fearful). 
	NOTE: Results from Bradford et al.7,25 using two separate threat uncertainty tasks have shown a pattern of results in self-reported anxiety which closely matched that of startle potentiation.</li>
	<li>Debrief the participant, compensate them for their time, and dismiss them.</li>
	<li>Clean and disinfect all sensors.</li>
</ol>
6. Data Processing, Reduction, and Analysis
NOTE: Researchers can accomplish data processing and reduction with various software packages. EEGLAB36 is a free, open source toolbox for analysis of psychophysiological data within Matlab37. For a template EEGLAB script of data processing and reduction steps please see the supplementary material. Data processing and reduction follow published guidelines27. For a display of a few seconds of the unprocessed (raw) continuous EMG signal surrounding one startle probe, see Figure 4A.
<ol>
	<li>Apply a forward-backward high pass filter (4th order 28 Hz Butterworth filter) to the raw continuous EMG (see Figure 4A,B).</li>
	<li>Rectify the filtered continuous EMG (see Figure 4C).</li>
	<li>Smooth the rectified EMG signal using a forward-backward 4th order 30 Hz Butterworth low pass filter (see Figure 4D).</li>
	<li>Epoch the smoothed continuous signal, retaining -50 to 250 msec surrounding the acoustic startle probe onset and &#8220;Baseline correct&#8221; the epoched signal by subtracting the mean of the pre-probe baseline (-50 to 0 msec) from the entire epoched signal (see Figure 4E).</li>
	<li>Score startle response from each epoch as maximum response between 20 and 100 msec post-probe onset (see Figure 4F).</li>
	<li>Reject trials with excessive artifact (e.g., excessive deflections in the pre-probe baseline; see Figure 5). 
	NOTE: Signals that contain greater than 40 &#956;V deflections in the pre-probe baseline may be identified as artifact.</li>
	<li>Average startle response for epochs within each task condition (no-shock, 20% shock, 100% shock) (see Figure 6A).
	<ol>
		<li>Calculate startle potentiation for uncertain shock as the difference between mean startle response to startle probes during 20% shock cues vs. no-shock cues (see Figure 6B). NOTE: Startle response to ITI probes during the 20% condition can also be measured to study effects of anticipation and sustained startle potentiation relevant to some conceptualizations of anxiety6,21.</li>
		<li>Calculate startle potentiation for certain shock as the difference between mean startle response to startle probes during 100% shock cues vs. no-shock cues (see Figure 6B).</li>
	</ol>
	</li>
	<li>Analyze startle potentiation using a General Linear Model with repeated measures on task condition and general startle reactivity (calculated in step 2.5) as an additive or interactive covariate32.</li>
</ol>

The authors declare that they have no competing financial interests.

The Threat Probability Task can be used to study the expression of anxiety and fear by assessing startle potentiation to low probability (uncertain) and high probability (certain) threat of electric shock. The primary dependent measure and threat contingencies used in this task can be used with rodents, non-human primates and humans, thus, providing an excellent translational tool for studying the expression of negative affect13,18,40. Startle potentiation to threat of electric shock has clear connections to defensive system activation, is resistant to volitional control, and has well defined neurobiological substrates. This is in contrast to self-report measures of affect that may be unduly influenced by demand characteristics and are distal to known neurobiology. The Threat Probability Task offers the precision to parse distinct forms of negative affect in contrasts to other common psychophysiological methods like the Emotional Picture Viewing task. The Threat Probability Task is straightforward to implement and easy to analyze in contrast to other more costly and technical methods such as fMRI.
The Threat Probability Task is one example of a broader class of threat tasks that manipulate threat uncertainty using cued electric shock as the threat and startle potentiation as the dependent measure. As such, there is considerable flexibility in cue-shock contingencies and shock characteristics that can support a program of research with strong conceptual replications. For example, the Threat Probability Task is a derivative of the previously validated No-Shock, Predictable Shock, Unpredictable shock task21. The NPU task manipulates uncertainty regarding both IF (shock probability) and WHEN (shock timing) shocks will occur. The NPU task has been used to examine drug administration and deprivation effects on negative affective response4,34 and etiological mechanisms in mood and anxiety disorders22&#8211;24,41&#8211;43. In other research, Curtin and colleagues have also developed variants of these cued threat tasks that precisely manipulate threat uncertainty about WHEN (shock timing)5,29,44; WHERE (administration location on body for shock)25; and HOW BAD (shock intensity)7. While Curtin et al.&#8217;s use of these tasks has thus far focused on the effects of drug administration and withdrawal in healthy participants; all of these tasks could be used to study anxiety and fear responses in patients with anxiety and other mental disorders2,45.
Curtin and colleagues have used all of the aforementioned classes of cued threat tasks in a program of research that has probed the boundary conditions of alcohol&#8217;s anxiolytic effects on anxiety expressed during uncertain threat, broadly defined. In all of these tasks, alcohol had a significantly greater stress response dampening effect on startle potentiation during uncertain than certain threat. The consistent pattern of results supports the validity of the threat uncertainty construct and the use of this whole class of tasks to manipulate this construct. Animal neuroscience addiction models suggest that neuroadaptation in the response to uncertain threats and other stressors following repeated, chronic drug use provides one important mechanism in the etiology of alcohol and other drug addiction8. Research from our laboratory using these cued threat tasks has provided preliminary support for this etiological mechanism in humans29,34,44.
Although startle potentiation provides an attractive translational measure of defensive reactivity to aversive events in the Threat Probability Task, cross-validation with other distinct measures of negative affect responding will reduce concerns about alternative explanations that could be specific to this dependent measure. In fact, the Threat Probability Task can easily accommodate other dependent measures. For example, event related potentials, prepulse inhibition of startle, and behavioral responses can be examined in the Threat Probability Task to allow researchers to probe differences in attentional function during anxiety versus fear15,17,46. Researchers interested in anxiety and fear acquisition, rather than expression, can modify the instructions and stimulus parameters in the Threat Probability Task to better serve their research questions. As noted earlier, retrospective self-report of anxiety/fear after each set or at the end of the task can be obtained easily7. Online measurement of perceived shock likelihood can also be obtained via keyboard press or voice recording to ensure that participants maintain attention and understanding of instructions throughout the task (for an example see47). Future work with the Threat Probability Task and similar cued threat tasks can combine other methods while using clinical patient populations to increase the external validity of the task and further define anxiety and fear. For example, it is possible to correlate individual differences in the responding during the Threat Probability Task with responding to threats and other stressors in the &#8220;real world&#8221; that are assessed via ecological momentary assessment (EMA). Furthermore, participant responding during the Threat Probability Task can be used as a surrogate endpoint48&#8211;50 to study the effects of pharmacological and/or behavioral interventions to treat mood and anxiety disorders and substance dependence disorders.
The Threat Probability Task can also accommodate threats other than electric shock as well as other startle measurement methods. For example, potentiation of the eyeblink startle response has been confirmed in response to aversive air blast directed at the throat51 and aversive loud noise52. The startle response can be potentiated by darkness in humans, which provides a clear translational bridge to light potentiated startle in rodents (a nocturnal species13). The eye-blink startle response can also be elicited by probes in other sensory modalities including visual27 and tactile53. Clearly, measurement of startle potentiation in the Threat Probability Task and related cued threat tasks provides a flexible tool to affective scientists interested in normative and pathological negative affective response.

The overall goal of the Threat Probability Task is to experimentally disentangle the expression of anxiety in response to low probability (i.e., uncertain) threats from fear in response to high probability (i.e., certain) threats. Uncertainty occurs when some aspect of a threat is poorly defined. While anxiety can be described in numerous ways, exacerbated responses to low probability or otherwise uncertain negative events is a distinguishing clinical symptom in anxiety disorders1,2. Furthermore, increased anxiety related physiological responding during uncertain threat of shock versus fear related physiological responding during certain threat of shock in laboratory tasks may provide a physiological marker for anxiety disorders3. Dampening of anxiety to uncertain threats specifically may be a critical component of the stress response dampening properties of drugs such as alcohol4-7. Increased anxiety during uncertain threat may mark a neuroadaptation in the brain&#8217;s stress circuitry following chronic drug use4,8. Thus, the Threat Probability Task provides an objective measure of negative affective and distinct emotional states (anxiety, fear) to use in research on psychopathology, substance use/abuse and affective science. As such, it can be a powerful tool for use by clinical and affective scientists interested in psychopathology etiology and individual differences in emotion.
Traditional methods used to study emotions in humans
Affective scientists have used numerous measures and paradigms to study human emotion9 but most of these do not provide the necessary precision found in the Threat Probability Task to parse anxiety from other negative emotions such as fear. For example, self-report is commonly used but it may suffer from demand characteristics and other forms of response bias. Participants may not be able to accurately distinguish between anxiety and fear, and the connection of their report to underlying neurobiological mechanisms is distal at best. Furthermore, self-report must often be conducted retrospectively since the process of introspection and report might otherwise alter participants&#8217; experience of the affective stimuli. Of course, retrospective report suffers from memory interference and degradation. Psychophysiologists often measure emotions during an affect manipulation that involves presentation of emotionally evocative pictures10. This picture viewing task is well validated, is less affected by the shortcomings of self-report, and has resulted in many important insights regarding individual differences in affective response and their contribution to psychopathology11,12. However, only broad negative affect is measured during this picture viewing task which does not allow for the study of distinct negative emotions such as anxiety and fear which can be measured with the Threat Probability Task. Affective neuroscientists frequently measure functional magnetic resonance imaging (fMRI) during tasks that elicit negative affect but these approaches may be too costly for many researchers. Furthermore, the spatial and temporal resolutions of fMRI methods are currently limited, making it difficult for fMRI to disentangle the neurological structures believed to be associated with anxiety versus other emotions. More importantly, a well-defined fMRI index of any type of negative affect has yet to be established.
Translational research with animals using the startle response
The Threat Probability Task is modeled after basic research with animals that provided the first example of the precision needed to disentangle anxiety from fear. Neuroscientists have used carefully controlled lesion studies with rodents to model anxiety and fear using differential responses to uncertain and certain cued threat of electric shock. This work has elucidated important differences in anxiety related responses to low probability, ambiguously defined, distal or otherwise uncertain shock versus fear related responses to highly probable, clearly defined, imminent certain shock13. Uncertain threats elicit freezing and hyper vigilance in animals, whereas certain threats elicit active avoidance, defensive attack, or both14. Imminent, certain threats focus attention on the threat itself, whereas distal, temporally uncertain threats encourage distributed attention to the overall environment15&#8211;17. Response to temporally uncertain threats appears to be sustained, whereas response to certain threats is phasic and time-locked to the threat13. In related work, lesion studies have shown that response to uncertain threats are selectively mediated by corticotrophin-releasing factor and norepinephrine pathways through the lateral divisions of the central nucleus of the amygdala and the bed nucleus of the stria terminalis18. Much of this work uses potentiation of the acoustic startle response as a primary dependent measure13, which is the same dependent measure used in the Threat Probability Task. The neurobiological substrates of the startle response circuit have been extensively studied with the discovery of clear connections to the brain structures active in responses to uncertain and certain threats19,20. The startle response can be assessed in numerous species which provides a powerful translational tool to study emotions. The startle response in humans occurs reflexively in response to a sudden and intense auditory stimulus. Startle is most often measured in humans by the placement of electromyography (EMG) electrodes on the orbicularis oculi (lid closing) muscle of the eye. Startle related EMG activity is potentiated when an organism is presented with a threatening stimulus such as an impending electric shock relative to non-threatening stimuli19.
The No-shock, Predictable-shock, Unpredictable-shock (NPU) task and threat uncertainty
The Threat Probability Task was inspired by Grillon and colleagues when these researchers introduced the use of startle potentiation to study anxiety and fear in humans with the No-shock, Predictable-shock, Unpredictable-shock (NPU) task21. In the Predictable condition of the NPU task, shocks are 100 percent cue-contingent and occur at a consistent, known time (end of brief cue presentation). In the Unpredictable condition of the NPU task, shocks are fully unpredictable. Patients with posttraumatic stress and panic disorders exhibit selectively increased startle potentiation during unpredictable but not predictable shock in the NPU task22,23. In other work, medications prescribed to treat anxiety have a greater effect on startle potentiation during unpredictable shock than during predictable shock in the NPU task24. In research on the anxiolytic effects of alcohol, Moberg and Curtin4 used the NPU task to demonstrate that a moderate dose of alcohol selectively reduces startle potentiation during threat of unpredictable but not predictable shock. Uncertainty is multifaceted and shocks in the unpredictable condition of the NPU task are uncertain in regards to both IF they are to occur (probability uncertainty) and WHEN they occur (temporal uncertainty). Many theories suggest that the WHEN dimension of uncertainty is critical in producing anxiety19. However, data from Curtin et al.5 suggests a common mechanism for the elicitation of anxiety across various types of uncertainty. The Threat Probability task described here manipulates uncertainty about IF a shock will occur while holding all other dimensions of uncertainty constant thus making clear what aspect of uncertainty is responsible for the effects the task presents. Tasks that use startle potentiation to cued threat are flexible and can also be modified by affective scientists to manipulate uncertainty about WHERE the shocks are going to occur25 and HOW BAD they will be7,26. Of all of these tasks, the Threat Probability Task is one of the easiest to interpret due to its focus on one dimension of uncertainty and most straightforward to implement due to its inclusion of only two threat uncertainty variants (low probability and high probability shock).
The Threat Probability Task
In the Threat Probability Task, the participant is seated approximately 1.5 m from a cathode ray tube (CRT) monitor. Threat cues are displayed on the monitor for 5 sec each with a variable duration ITI (range = 15-20 sec). Threat cues are divided into sets of two shock threat conditions and one no-threat condition (see Figure 1). In both threat conditions shocks of 200 msec duration are delivered at 4.5 sec into cue presentation times to the participant&#8217;s fingers. In the 100% threat probability condition, shocks are delivered during presentation of every cue. In 20% threat probability condition, shocks are delivered during presentation of 1 out of every 5 cues. The participant sees two sets (15 cues total) of each threat probability condition. The participant also sees two neutral sets of cues that signal no threat (no-threat cues; 15 cues total). Text displayed on the monitor informs the participant of the next set type. A label for the set type is displayed during the entire set in the upper left corner of the monitor. Different color cues are used for each condition to facilitate awareness of each set for the participant. Throughout the task, the stimulus presentation program presents the participant with acoustic startle probes in the form of 50 msec bursts of 102 dB white noise with near instantaneous rise time delivered through headphones. Acoustic startle probes are delivered at 4 sec into the presentation of a subset of the cues. Additional probes are delivered at 13 sec and 15 sec post cue offset during the ITIs to decrease the predictability of the probes. Before any presentation of visual stimuli, the task begins with the delivery of 3 acoustic startle probes to habituate the startle response immediately before main task measurement. Researchers balance the serial position of the acoustic startle probes across conditions within subjects in order to control for habituation and sensitization effects27,28. For an example of one fully counterbalanced series of trials for the Threat Probability Task see Supplementary Material.
The Threat Probability task has been used to demonstrate that low probability (uncertain) shock alone is sufficient to elicit anxiety and allow assessment of the anxiolytic effects of alcohol6. Preliminary research with dependent marijuana users suggests the Threat Probability Task can also be used to assess the effects of drug withdrawal29. Thus, the Threat Probability Task provides an easily implemented alternative to more expensive and less precise methods for the objective measure of distinct negative emotional states (e.g., anxiety and fear) for research on psychopathology, substance use/abuse, and broad affective science.

<table><tbody><tr><td>Amplifier</td><td>Numerous options</td><td></td><td>See Curtin, Lorenzo, and Allen (2007) for a list of vendors.</td></tr><tr><td>Small Ag/AgCl EMG Sensors</td><td><a href="http://www.discountdisposables.com/" target="_blank"><img src="/img/new_window.png" /> Discount Disposables</a></td><td>TDE-023-Y-ZZ-S</td><td>4 mm, and 48 in lead length</td></tr><tr><td>Large Ag/AgCl EMG sensor</td><td><a href="http://www.discountdisposables.com/" target="_blank"><img src="/img/new_window.png" /> Discount Disposables</a></td><td>TDE-022-Y-ZZ-S</td><td>8 mm, and 48 in lead length</td></tr><tr><td>Small electrode collars</td><td><a href="http://www.discountdisposables.com/" target="_blank"><img src="/img/new_window.png" /> Discount Disposables</a></td><td>TD-23</td><td>5 mm</td></tr><tr><td>Large electrode collars</td><td><a href="http://www.discountdisposables.com/" target="_blank"><img src="/img/new_window.png" /> Discount Disposables</a></td><td>TD-22</td><td>8 mm</td></tr><tr><td>Shock box</td><td>Custom</td><td>Custom</td><td>See supplemental material for a circuit diagram for the custom shock box used by the Curtin laboratory. An example of a commerical shock box can be found at: &lt;a href=&quot;http://www.psychlab.com/stim_SHK_shockers.html&quot;&gt;http://www.psychlab.com/stim_SHK_shockers.html&lt;/a&gt;.</td></tr><tr><td>Alcohol pads</td><td><a href="http://www.fishersci.com/ecomm/servlet/home?storeId=10652" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific</a></td><td>06-669-72</td><td></td></tr><tr><td>Exfoliant gel</td><td><a href="http://www.weaverandcompany.com/index.html" target="_blank"><img src="/img/new_window.png" /> Weaver and Company</a></td><td>NuPrep</td><td></td></tr><tr><td>Conductive Gel</td><td><a href="http://www.electro-cap.com/" target="_blank"><img src="/img/new_window.png" /> Electro-Cap International</a></td><td>ECA E9</td><td></td></tr><tr><td>Gauze pads</td><td><a href="http://www.neuroscan.com/supplies.cfm" target="_blank"><img src="/img/new_window.png" /> Neuromedical Supplies</a></td><td>95000025</td><td></td></tr><tr><td>Blunt Needle</td><td><a href="http://www.electro-cap.com/" target="_blank"><img src="/img/new_window.png" /> Electro-Cap International</a></td><td>E8B</td><td></td></tr><tr><td>Medical tape</td><td><a href="http://www.neuroscan.com/supplies.cfm" target="_blank"><img src="/img/new_window.png" /> Neuromedical Supplies</a></td><td>95000032</td><td></td></tr><tr><td>Electrode Sterilizing Solution</td><td><a href="http://www.buyemp.com" target="_blank"><img src="/img/new_window.png" /> Emergency Medical Products:</a></td><td>MX-2800</td><td>Gloves should be warn when handling metricide.</td></tr><tr><td>Headphones</td><td><a href="http://en-us.sennheiser.com/" target="_blank"><img src="/img/new_window.png" /> Sennheiser</a></td><td>4974</td><td>Head phones should be capable of repeatedly delivering startle probe&amp;rsquo;s at the level chosen by experimenters (&lt;em&gt;e.g.&lt;/em&gt;102 dB).</td></tr><tr><td>Participant monitoring camera</td><td><a href="http://www.polaris.com/en-us/home.aspx" target="_blank"><img src="/img/new_window.png" /> PolarisUSA</a></td><td>BC-660B</td><td>Infrared capable camera so participant can be monitored while lights are off in experiment room.</td></tr><tr><td>Infrared panel</td><td>PolarisUSA</td><td>IR-TILE</td><td>http://www.polaris.com/en-us/home.aspx</td></tr><tr><td>Video monitor for participant monitoring</td><td><a href="http://www.marshall-usa.com/IVS/monitors/M-Pro_CCTV_19.html" target="_blank"><img src="/img/new_window.png" /> Marshall Electronics</a></td><td>M-Pro CCTV 19</td><td></td></tr><tr><td>Stimulus Computer</td><td><a href="http://www.dell.com/" target="_blank"><img src="/img/new_window.png" /> Dell</a></td><td>Dell Optiplex3010</td><td>Most modern computers appropriate</td></tr><tr><td>Sound card (Stimulus computer)</td><td><a href="http://us.store.creative.com/Creative-Sound-Blaster-XFi-Titanium-HD/M/B0041OUA38.htm" target="_blank"><img src="/img/new_window.png" /> Creative</a></td><td>70SB127000002</td><td>The sound card delivers the startle probes. An example of a stand alone noise generator can be found at: &lt;a href=&quot;http://www.psychlab.com/stim_TG_WN_sound.html#&quot;&gt;http://www.psychlab.com/stim_TG_WN_sound.html#&lt;/a&gt;.</td></tr><tr><td>I/O card (Stimulus computer)</td><td><a href="http://www.mccdaq.com/pci-data-acquisition/PCI-DIO24.aspx" target="_blank"><img src="/img/new_window.png" /> Measurement Computing</a></td><td>PCI-DIO24</td><td>I/O card allows control of shock box and communication of event markers (&lt;em&gt;e.g.&lt;/em&gt; for startle probe occurrence) to data collection computer.</td></tr><tr><td>Stimulus control software</td><td><a href="http://www.psychtoolbox.org/" target="_blank"><img src="/img/new_window.png" /> Psychtoolbox</a></td><td></td><td>Open source (free) toolbox based in Matlab.</td></tr><tr><td>Computational platform for stimulus control and data reduction</td><td><a href="http://www.mathworks.com/products/matlab/" target="_blank"><img src="/img/new_window.png" /> MathWorks</a></td><td></td><td>Required to use Psychtoolbox and EEGLAB (below).</td></tr><tr><td>Data collection computer</td><td><a href="http://www.dell.com/" target="_blank"><img src="/img/new_window.png" /> Dell</a></td><td>Dell Optiplex3010</td><td>Most modern computers are appropriate</td></tr><tr><td>Psychophysiology acquisition software</td><td>Numerous options</td><td></td><td>See Curtin, Lorenzo, and Allen (2007) for a list of vendors.</td></tr><tr><td>Stimulus Monitor</td><td><a href="http://us.acer.com/ac/en/US/content/group/monitors" target="_blank"><img src="/img/new_window.png" /> Acer</a></td><td>Acer AL1916W</td><td></td></tr><tr><td>Data Collection Monitor</td><td><a href="http://us.acer.com/ac/en/US/content/group/monitors" target="_blank"><img src="/img/new_window.png" /> Acer</a></td><td>Acer AL1916W</td><td></td></tr><tr><td>Participant CRT monitor</td><td><a href="http://www.viewsonic.com/us/" target="_blank"><img src="/img/new_window.png" /> ViewSonic</a></td><td>P810</td><td></td></tr><tr><td>Data processing software</td><td><a href="http://sccn.ucsd.edu/eeglab/" target="_blank"><img src="/img/new_window.png" /> EEGLAB</a></td><td></td><td>Open source (free) software package based in Matlab.</td></tr></tbody></table>

using the threat probability task to assess anxiety and fear during uncertain and certain threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., &#8220;startle probes&#8221;). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.

The Threat Probability Task produces robust startle potentiation during both 100% (certain) probability and 20% (uncertain) probability threat cues (see Figure 6B). Previous results using this task show startle potentiation during the uncertain (20%) threat condition to be significantly increased above startle potentiation during high probability (100%) (certain) threat condition. Acute administration of a moderate does of alcohol (target blood alcohol concentration of 0.08%) produces selectively greater reduction in startle potentiation during 20% (uncertain) threat versus 100% (certain) threat (see Figure 7) in humans. This confirms the &#8220;classic&#8221; stress response dampening effect of alcohol on anxiety38,39. Similarly, short term (3 day) deprivation of marijuana among heavy daily marijuana users produces selective greater increase in startle potentiation during 20% (uncertain) threat vs. 100% (certain) threat in humans (see Figure 8). This result is consistent with current rodent neuroscience evidence that implicates selective &#8220;stress neuroadaptation&#8221; in brain circuits responsible for anxiety during uncertain threats or other stressors8,13.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig1highres.jpg" width="500" /> 
Figure 1. Cues in the Threat Probability Task are divided into two threat of shock (shocks displayed with yellow lightning bolts) probability conditions of 100% probability and 20% probability. Shocks occur 4.5 sec into the cue presentation times in threat blocks. Startle potentiation to the threat cues is calculated from startle to sets of no-threat cues. Each cue type is displayed in a different color to facilitate understanding of the current condition. Acoustic startle probes (displayed as black asterisks) are presented 4 sec into the cue presentation times. Acoustic startle probes are also presented at 13 or 15 sec into the ITI periods between the cues.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig2highres.jpg" width="300" /> 
Figure 2. EMG electrode placement. Two small (4 mm) electro-conductive gel filled EMG Ag-AgCl electrodes are placed over the lower portion of the orbicularis oculi muscle. One large (8 mm) Ag-AgCl ground electrode is placed on non-focal site such as the participant&#8217;s forehead.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig3highres.jpg" width="600" /> 
Figure 3. Raw EMG signals. Panel A displays 2 sec of the EMG signal surrounding presentation of a startle probe (vertical gray dotted line). Panel B displays a similar period of EMG signal that is contaminated by a high degree of electrical noise due to high impedances. Panel C displays a similar period of EMG signal that is contaminated by movement artifact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig3highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig4highres.jpg" width="600" /> 
Figure 4. Data Reduction for Startle Response. Panels A-F display a raw EMG signal taken through each processing step described in the protocol. The occurrence of the startle probe is indicated by a vertical gray dotted line) Panel A displays an unprocessed EMG signal. Panel B Displays an EMG signal that has been high pass filtered to remove low frequency artifact. Panel C displays the same EMG signal after rectification. Panel D displays the same EMG signal after it has been smoothed by a low pass filter. Panel E displays the same EMG signal after it has been epoched and baseline corrected. Panel F displays the same EMG signal as panel E with the gray band representing the 20 msec to 100 msec scoring window for peak startle response (marked with a vertical hatch mark). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig4highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig5highres.jpg" width="300" /> 
Figure 5. Excessive artifact in pre-probe baseline. A fully processed EMG signal with excessive pre-probe baseline activity. In this case, the participant moved or blinked immediately before the acoustic startle probe presentation (vertical gray dotted line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig5highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig6highres.jpg" width="600" /> 
Figure 6. Trial level startle and mean startle potentiation by condition. Panel A shows one fully processed individual trial from each of the three conditions (20% shock [in blue], 100% shock [in red], No Shock [in green]) superimposed on one another. Presentation of the startle probe is indicated by a vertical gray dotted line. The gray band represents the 20 msec to 100 msec scoring window for peak startle response. Peak scored startle response is indicated by a vertical hatch line. Panel B shows the average startle potentiation after subtracting the mean scored response across the 6 individual trials in the No-Shock condition from the mean scored response across the 8 individual trials for each shock condition. Mean startle potentiation for the 100% shock and 20% shock conditions are displayed in red and blue, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig6highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig7highres.jpg" width="400" /> 
Figure 7. Startle potentiation by Beverage group and threat probability. Mean startle potentiation for the 20% shock condition is displayed with the blue line. Mean startle potentiation for the 100% shock condition is displayed with the red line. Standard error of the mean from point estimates of predicted startle potentiation in the General Linear Model are displayed with error bars6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig7highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/51905/51905fig8highres.jpg" width="400" /> 
Figure 8. Startle potentiation by Marijuana group and threat probability. Mean startle potentiation for the 20% shock condition is displayed with the blue line. Mean startle potentiation for the 100% shock condition is displayed with the red line. Standard error of the mean from point estimates of predicted startle potentiation in the General Linear Model are displayed with error bars29. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/51905fig8highres.jpg" target="_blank">Click here to view a larger version of this figure.</a>
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/SupplimentalFigure1.pdf" target="_blank">Supplemental Figure 1.</a> Circuit diagram for the custom shock box used in John Curtin&#8217;s Addiction Research Laboratory.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51905/Table1_51905.xlsx">Supplemental Table 1.</a> A fully counterbalanced series of trials for the Threat Probability Task. Multiple orders should be used across participants to reduce order effects.

Watch this Scientific Journal Video about Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat at JoVE.com

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

using-threat-probability-task-to-assess-anxiety-fear-during-uncertain

Nanyang Technological University

Research

JoVE Journal

Behavior

15.1K Views.  University of Wisconsin-Madison. Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science. 

Video: Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic &#946;-Blocker

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a &#946;-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.

Disrupting reconsolidation is a promising approach to dampen the behavioral expression of fear memory in patients with anxiety disorders or posttraumatic stress disorder. In a series of human fear conditioning studies we showed that disrupting reconsolidation by the noradrenergic &#946;-blocker propranolol is very effective in erasing conditioned fear responding.

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive ...

Measuring Attentional Biases for Threat in Children and Adults

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this domain has focused almost exclusively on adult participants, completely ignoring the topic of threat detection over the course of development. One of the biggest reasons for the lack of developmental work in this area is likely the absence of a reliable paradigm that can measure perceptual biases for threat in children. To address this issue, we recently designed a modified visual search paradigm similar to the standard adult paradigm that is appropriate for studying threat detection in preschool-aged participants. Here we describe this new procedure. In the general paradigm, we present participants with matrices of color photographs, and ask them to find and touch a target on the screen. Latency to touch the target is recorded. Using a touch-screen monitor makes the procedure simple and easy, allowing us to collect data in participants ranging from 3 years of age to adults. Thus far, the paradigm has consistently shown that both adults and children detect threatening stimuli (e.g., snakes, spiders, angry/fearful faces) more quickly than neutral stimuli (e.g., flowers, mushrooms, happy/neutral faces). Altogether, this procedure provides an important new tool for researchers interested in studying the development of attentional biases for threat.

Here we describe a touch-screen visual search paradigm that can be used to study threat detection across the lifespan. The paradigm has already been used in various studies demonstrating that both children and adults detect threatening stimuli like snakes, spiders, and angry faces faster than non-threatening stimuli.

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Medicine

Experimental Research Examining How People Can Cope with Uncertainty Through Soft Haptic Sensations

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has focused on the cognitive strategies people adopt to deal with uncertainty. However, especially when uncertainty is due to unpredictable societal events (e.g., economical crises, political revolutions, terrorism threats) of which one is unable to judge the impact on one&#8217;s future live, cognitive strategies (like seeking additional information) is likely to fail to combat uncertainty. Instead, the current paper discusses a method demonstrating that people might deal with uncertainty experientially through soft haptic sensations. More specifically, because touching something soft creates a feeling of comfort and security, people prefer objects with softer as compared to harder properties when feeling uncertain. Seeking for softness is a highly efficient and effective tool to deal with uncertainty as our hands are available at all times. This protocol describes a set of methods demonstrating 1) how environmental (un)certainty can be situationally activated with an experiential priming procedure, 2) that the quality of the softness experience (what type of softness and how it is experienced) matters and 3) how uncertainty can be reduced using different methods.

To date research has focused on cognitive strategies people adopt to cope with uncertainty. This research examines instead an experiential way of dealing with uncertainty and introduces a set of experimental methods showing how the experience of haptic softness can serve as a tool to deal with uncertainty.

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has ...

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

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.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

Neuroscience

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

Threat-Safety Test for Stress Susceptibility or Resilience in Mice Models

Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience and its behavioral impact on mice should be comparable to humans. Chronic social defeat (CSD) utilizes a type of social stress involving physical attacks and sensory threats to induce mental dysfunctions resembling human affective disorders. To strengthen the psychosocial component of CSD, a 10-day CSD protocol was applied in which daily physical attacks are standardized to three 10 s episodes followed by a 24 h sensory phase. After the 10th sensory phase, the CSD protocol is followed by a refined behavioral assay called the social threat-safety test (STST). Post-stress behavioral assays need to determine how and to what extent the social stressor has influenced behavior. The STST allows chronically socially defeated male mice to interact with 2 novel male individuals (social targets): one social target from the attacking strain encountered during the CSD days and the other from a novel strain. Both are presented simultaneously in different compartments of a three-chambered test arena. The test enables a simultaneous assessment of social avoidance development to measure successful aversive conditioned learning and social threat-safety discrimination ability. The development of social avoidance towards both strains reflects a generalized aversive response and thus, a measurement of stress susceptibility. Meanwhile, the development of social avoidance towards only the attacking strain reflects threat-safety discrimination and thus, a measurement of stress resilience. Finally, the absence of social avoidance towards the attacking strain reflects impaired aversive conditioned learning. The protocol aims to refine the currently used mouse models of stress susceptibility/resilience by including translational criteria, specifically threat-safety discrimination and aversive response generalization, to categorize a single group of chronically socially defeated animals into resilient and susceptible subgroups, eventually advancing future translational approaches.

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience ...