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 d...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:216px;">Amplifier</td>
			<td rowspan="2" style="width:71px;">Numerous options</td>
			<td rowspan="2" style="width:119px;">N/A</td>
			<td rowspan="2" style="width:167px;">See Curtin, Lorenzo, and Allen (2007) for a list of vendors.&#160;&#160;</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="42">
			<td height="85" rowspan="2" style="height:85px;width:216px;">Small Ag/AgCl EMG Sensors</td>
			<td rowspan="2" style="width:71px;">Discount Disposables</td>
			<td rowspan="2" style="width:119px;">TDE-023-Y-ZZ-S</td>
			<td style="width:167px;">4mm, and 48in lead length</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.discountdisposables.com/</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="2" style="height:85px;width:216px;">Large Ag/AgCl EMG sensor</td>
			<td rowspan="2" style="width:71px;">Discount Disposables</td>
			<td rowspan="2" style="width:119px;">TDE-022-Y-ZZ-S</td>
			<td style="width:167px;">8mm, and 48in lead length</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.discountdisposables.com/</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="2" style="height:64px;width:216px;">Small electrode collars</td>
			<td rowspan="2" style="width:71px;">Discount Disposables</td>
			<td rowspan="2" style="width:119px;">TD-23</td>
			<td style="width:167px;">5mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.discountdisposables.com/</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="2" style="height:64px;width:216px;">Large electrode collars</td>
			<td rowspan="2" style="width:71px;">Discount Disposables</td>
			<td rowspan="2" style="width:119px;">TD-22&#160;&#160;</td>
			<td style="width:167px;">8mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.discountdisposables.com/</td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:216px;">Shock box</td>
			<td style="width:71px;">Custom</td>
			<td style="width:119px;">Custom</td>
			<td style="width:167px;">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: http://www.psychlab.com/stim_SHK_shockers.html&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Alcohol pads</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">06-669-72</td>
			<td style="width:167px;">http://www.fishersci.com/ecomm/servlet/home?storeId=10652</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Exfoliant gel</td>
			<td style="width:71px;">Weaver and Company</td>
			<td style="width:119px;">NuPrep</td>
			<td style="width:167px;">http://www.weaverandcompany.com/index.html</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Conductive Gel</td>
			<td style="width:71px;">Electro-Cap International</td>
			<td style="width:119px;">ECA E9</td>
			<td style="width:167px;">http://www.electro-cap.com/</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Gauze pads</td>
			<td style="width:71px;">Neuromedical Supplies</td>
			<td align="right" style="width:119px;">95000025</td>
			<td style="width:167px;">http://www.neuroscan.com/supplies.cfm</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Blunt Needle</td>
			<td style="width:71px;">Electro-Cap International</td>
			<td style="width:119px;">E8B</td>
			<td style="width:167px;">http://www.electro-cap.com/</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Medical tape</td>
			<td style="width:71px;">Neuromedical Supplies</td>
			<td align="right" style="width:119px;">95000032</td>
			<td style="width:167px;">http://www.neuroscan.com/supplies.cfm</td>
		</tr>
		<tr height="42">
			<td height="64" rowspan="2" style="height:64px;width:216px;">Electrode Sterilizing Solution</td>
			<td rowspan="2" style="width:71px;">Emergency Medical Products:</td>
			<td rowspan="2" style="width:119px;">MX-2800</td>
			<td style="width:167px;">Gloves should be warn when handling metricide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">http://www.buyemp.com</td>
		</tr>
		<tr height="126">
			<td height="169" rowspan="2" style="height:169px;width:216px;">Headphones</td>
			<td rowspan="2" style="width:71px;">Sennheiser</td>
			<td align="right" rowspan="2" style="width:119px;">4974</td>
			<td style="width:167px;">Head phones should be capable of repeatedly delivering startle probe&#8217;s at the level chosen by experimenters (e.g.,102 db)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://en-us.sennheiser.com/</td>
		</tr>
		<tr height="84">
			<td height="127" rowspan="2" style="height:127px;width:216px;">Participant monitoring camera.</td>
			<td rowspan="2" style="width:71px;">PolarisUSA</td>
			<td rowspan="2" style="width:119px;">BC-660B</td>
			<td style="width:167px;">Infrared capable camera so participant can be monitored while lights are off in experiment room.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.polaris.com/en-us/home.aspx</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Infrared panel</td>
			<td style="width:71px;">PolarisUSA</td>
			<td style="width:119px;">IR-TILE</td>
			<td style="width:167px;">http://www.polaris.com/en-us/home.aspx</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Video monitor for participant monitoring</td>
			<td style="width:71px;">Marshall Electronics</td>
			<td style="width:119px;">M-Pro CCTV 19</td>
			<td style="width:167px;">http://www.marshall-usa.com/IVS/monitors/M-Pro_CCTV_19.html</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:216px;">Stimulus Computer</td>
			<td rowspan="3" style="width:71px;">Dell</td>
			<td rowspan="3" style="width:119px;">Dell Optiplex3010&#160;</td>
			<td style="width:167px;">Most modern computers appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">http://www.dell.com/</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:216px;">Sound card (Stimulus computer)</td>
			<td style="width:71px;">Creative</td>
			<td style="width:119px;">70SB127000002</td>
			<td style="width:167px;">http://us.store.creative.com/Creative-Sound-Blaster-XFi-Titanium-HD/M/B0041OUA38.htm. The sound card delivers the startle probes. An example of a stand alone noise generator can be found at: http://www.psychlab.com/stim_TG_WN_sound.html#</td>
		</tr>
		<tr height="126">
			<td height="190" rowspan="2" style="height:190px;width:216px;">I/O card (Stimulus computer)</td>
			<td rowspan="2" style="width:71px;">Measurement Computing</td>
			<td rowspan="2" style="width:119px;">PCI-DIO24</td>
			<td style="width:167px;">I/O card allows control of shock box and communication of event markers (e.g., for startle probe occurrence) to data collection computer.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:167px;">http://www.mccdaq.com/pci-data-acquisition/PCI-DIO24.aspx</td>
		</tr>
		<tr height="42">
			<td height="64" rowspan="2" style="height:64px;width:216px;">Stimulus control software</td>
			<td rowspan="2" style="width:71px;">Psychtoolbox</td>
			<td rowspan="2" style="width:119px;">N/A</td>
			<td style="width:167px;">Open source (free) toolbox based in Matlab</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Psychtoolbox.org</td>
		</tr>
		<tr height="63">
			<td height="106" rowspan="2" style="height:106px;width:216px;">Computational platform for stimulus control and data reduction</td>
			<td rowspan="2" style="width:71px;">MathWorks</td>
			<td rowspan="2" style="width:119px;">N/A</td>
			<td style="width:167px;">Required to use Psychtoolbox and EEGLab (below)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://www.mathworks.com/products/matlab/</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:216px;">Data collection computer</td>
			<td rowspan="3" style="width:71px;">Dell</td>
			<td rowspan="3" style="width:119px;">Dell Optiplex3010</td>
			<td style="width:167px;">Most modern computers are appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">http://www.dell.com/</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Psychophysiology acquisition software</td>
			<td style="width:71px;">Numerous options</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">See Curtin, Lorenzo, and Allen (2007) for a list of vendors.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Stimulus Monitor</td>
			<td style="width:71px;">Acer</td>
			<td style="width:119px;">Acer AL1916W</td>
			<td style="width:167px;">http://us.acer.com/ac/en/US/content/group/monitors</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Data Collection Monitor</td>
			<td style="width:71px;">Acer</td>
			<td style="width:119px;">Acer AL1916W</td>
			<td style="width:167px;">http://us.acer.com/ac/en/US/content/group/monitors</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Participant CRT monitor</td>
			<td style="width:71px;">ViewSonic</td>
			<td style="width:119px;">P810</td>
			<td style="width:167px;">http://www.viewsonic.com/us/</td>
		</tr>
		<tr height="63">
			<td height="106" rowspan="2" style="height:106px;width:216px;">Data processing software</td>
			<td rowspan="2" style="width:71px;">EEGLab</td>
			<td rowspan="2" style="width:119px;">N/A</td>
			<td style="width:167px;">Open source (free) software package based in Matlab</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">http://sccn.ucsd.edu/eeglab/</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...

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

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

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

fMRI Validation of fNIRS Measurements During a Naturalistic Task

We present a method to compare brain activity recorded with near-infrared spectroscopy (fNIRS) in a dance video game task to that recorded in a reduced version of the task using fMRI (functional magnetic resonance imaging). Recently, it has been shown that fNIRS can accurately record functional brain activities equivalent to those concurrently recorded with functional magnetic resonance imaging for classic psychophysical tasks and simple finger tapping paradigms. However, an often quoted benefit of fNIRS is that the technique allows for studying neural mechanisms of complex, naturalistic behaviors that are not possible using the constrained environment of fMRI. Our goal was to extend the findings of previous studies that have shown high correlation between concurrently recorded fNIRS and fMRI signals to compare neural recordings obtained in fMRI procedures to those separately obtained in naturalistic fNIRS experiments. Specifically, we developed a modified version of the dance video game Dance Dance Revolution (DDR) to be compatible with both fMRI and fNIRS imaging procedures. In this methodology we explain the modifications to the software and hardware for compatibility with each technique as well as the scanning and calibration procedures used to obtain representative results. The results of the study show a task-related increase in oxyhemoglobin in both modalities and demonstrate that it is possible to replicate the findings of fMRI using fNIRS in a naturalistic task. This technique represents a methodology to compare fMRI imaging paradigms which utilize a reduced-world environment to fNIRS in closer approximation to naturalistic, full-body activities and behaviors. Further development of this technique may apply to neurodegenerative diseases, such as Parkinson&#8217;s disease, late states of dementia, or those with magnetic susceptibility which are contraindicated for fMRI scanning.

We present a method to compare functional brain activity recorded during a naturalistic task using fNIRS with activity recorded during fMRI.

We present a method to compare brain activity recorded with near-infrared spectroscopy (fNIRS) in a dance video game task to that recorded in a reduced ...

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 ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

Using Gold-standard Gait Analysis Methods to Assess Experience Effects on Lower-limb Mechanics During Moderate High-heeled Jogging and Running

A limited number of studies have explored lower-limb biomechanics during high-heeled jogging and running, and most studies have failed to clarify the wearing experience of subjects. This protocol describes the differences in lower-limb kinematics and ground reaction force (GRF) between experienced wearers (EW) and inexperienced wearers (IEW) during moderate high-heeled jogging and running. A three-dimensional (3D) motion analysis system with a configured force platform was used to synchronously capture lower-limb joint movements and GRF. 36 young females volunteered to participate in this study and were asked about high-heeled shoe-wearing experience, including frequency, duration, heel types, and heel heights. Eleven who had the experience of 3 to 6 cm heels for a minimum of three days per week (6 h per day) for at least two years and eleven who wore high heels less than twice per month participated. Subjects performed jogging and running at comfortable low and high speeds, respectively, with the right foot completely stepping onto a force platform when passing by along a 10 m walkway. EW and IEW adopted different biomechanical adaptations while jogging and running. IEW exhibited a generally larger range of joint movement, while EW showed a dramatically larger loading rate of GRF during running. Hence, further studies on the lower-limb biomechanics of high-heeled gait should strictly control the wearing experience of the subjects.

This study investigated lower-limb kinematics and ground reaction force (GRF) during moderate high-heeled jogging and running. Subjects were divided into groups of experienced wearers and inexperienced wearers. A three-dimensional motion analysis system with a configured force platform captured lower-limb joint movements and GRF.

A limited number of studies have explored lower-limb biomechanics during high-heeled jogging and running, and most studies have failed to clarify the ...

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 ...

Continuous Theta Burst Stimulation of the Posterior Medial Frontal Cortex to Experimentally Reduce Ideological Threat Responses

Decades of behavioral science research have documented functional shifts in attitudes and ideological adherence in response to various challenges, but little work to date has illuminated the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to experimentally assess the causal contribution of cortical regions to threat-related ideological shifts. In the example protocol provided here, participants are exposed to a threat prime-an explicit reminder of their own inevitable death and bodily decomposition-following a downregulation of the posterior medial frontal cortex (pMFC) or a sham stimulation. Next, disguised within a series of distracter tasks, participants' relative degree of ideological adherence is assessed-in the present example, with regard to coalitional prejudice and religious belief. Participants for whom the pMFC has been downregulated exhibit less coalitionally biased responses to an immigrant critical of the participants' national in-group, and less conviction in positive afterlife beliefs (i.e., God, angels, and heaven), despite having recently been reminded of death. These results complement prior findings that continuous theta burst stimulation of the pMFC influences social conformity and sharing and illustrate the feasibility of investigating the neural basis of high-level social cognitive shifts using transcranial magnetic stimulation.

Threats reliably evoke shifts in high-level ideological investment, but little work to date has explored the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to test the contribution of the posterior medial frontal cortex (and/or other regions) to threat-related ideological shifts.

Decades of behavioral science research have documented functional shifts in attitudes and ideological adherence in response to various challenges, but ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...