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 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.
According to the Attention Control Theory (ACT), anxiety interferes with cognitive processing by competing for access to limited Working Memory (WM) resources1. However, the ACT does not address the inverse of this relationship (i.e. the effect of cognitive processing on anxiety). By manipulating anxiety during cognitive tasks using the threat of shock paradigm, it is possible to assess both the effect of anxiety on cognition and the effect of cognition on anxiety2,3,4,5. The purpose of this protocol is to demonstrate how to administer the Sternberg WM paradigm during threat of shock paradigm to probe the bi-directional relationship between anxiety and WM maintenance.
The threat of shock paradigm is widely used in the laboratory to manipulate state anxiety6,7,8,9,10,11 and can be implemented in healthy subjects2,3,4,5 and patients12,13,14,15 alike (see Bradford et al.16 for an example). The paradigm consists of alternating blocks of threat and safety17. Subjects are at risk of receiving unpredictable electrical stimulations during the threat blocks, but not during the safe blocks. Subjects' anxiety can be probed periodically using the acoustic startle reflex18,19. Subjects typically show larger startle responses during the threat blocks compared to the safe blocks, and this Anxiety-Potentiated Startle (APS) can be used as a peripheral index of change in the ongoing anxiety during the test17,18. Potentiated startle in the threat of shock paradigm is recognized by the National Institute of Mental Health (NIMH) as a physiological index of anxiety in their Research Domain Criteria matrix20. However, it is also possible to probe an individual's anxiety using a self-report Likert-type scale. Because threat of shock is a passive paradigm, other cognitive tasks can be conducted simultaneously21. By combining the threat of shock with the Sternberg WM task, it is possible to probe anxiety during WM maintenance3.
During the Sternberg WM paradigm, subjects are required to encode a series of letters in WM and respond after a brief interval3,22. Unlike more complex WM tasks (e.g., the N-back task)4,5,23, the Sternberg task does not require the manipulation of information in the WM3,22. In addition, subjects encode, maintain, and respond to items during distinct intervals. Together, these features make it possible to dissociate WM maintenance from other, more complex cognitive processes24. By probing APS during the WM maintenance interval, it is possible to determine the effect of WM maintenance on anxiety. Likewise, by comparing WM accuracy and Reaction Time (RT) between the threat and safe blocks, it is possible to determine the effect of anxiety on WM maintenance. This protocol will detail the procedural steps necessary to conduct the Sternberg WM paradigm during threat of shock, as well as the analytical steps necessary to assess APS, accuracy, and reaction time during the task.
All participants gave written informed consent approved by the National Institute of Mental Health (NIMH) Combined Neuroscience Institutional Review Board (IRB) and were compensated for participating.
1. Set Up the Equipment
NOTE: Set up the equipment as described below (see Figure 1A)3.
2. Program the Experiment Using Available Software
NOTE: A neurobehavioral systems software (here referred to as the experimental software; see the Table of Materials) was used. Other equivalent software can be used.
3. Run the Experiment
4. Analyze the Performance
NOTE: Analyze the performance data for a single participant using the following instructions.
5. Analyze Startle
6. Analyze the Self-report Data
This protocol yields three primary data types: accuracy, RT, and APS. For accuracy and RT, this protocol involves two experimental manipulations, threat and load. For accuracy, typical results show a main effect of load but no main effect of threat and no load-by-threat interaction (trials (F(1,18) = 84.34; p <0.01; see Figure 5). Subjects are typically more accurate on the low-load than the high-load trials. For RT, typical results show a main effect of both load (F(1,18) = 19.49; p <0.01) and threat (F(1,18) = 8.03; p = 0.01) but no load-by-threat interaction (see Figure 6). Subjects typically show faster RTs during low-load trials than during high-load trials and faster RTs during threat blocks than during safe blocks.
This protocol also involves two experimental manipulations for APS: load and startle timing. Typical results show a load-by-timing interaction (F(1,18) = 16.63; p <0.01; see Figure 7). Subjects typically show significantly larger APS during low load vs. high load trials, but only when the startle probe is delivered during the maintenance interval (MNT; maintenance period: t(18) = 3.92; p <0.01; ITI: p >0.05; d = 0.72). It should be noted that, because inferential statistics can vary from study to study, it is important to replicate these effects. After this experiment, a consistent decrease in APS as a function of task difficulty was found. This finding was observed in a verbal N-back task (3-back > 0-back d(25) = 2.2)4, the Sternberg WM paradigm (see above, d(18) = 0.72; for replication, see Experiment 1 in Balderston et al. 20163; high load > low load, d(18) = 0.44), and a complex picture recognition task (retrieval > encoding, d(21) = 0.47)2. However, it should be noted that the final result may be driven in part by habituation.
Although it is difficult to determine an individual's subjective affective state during each trial, self-report data can be used to determine the effectiveness of the anxiety manipulation and as an individual difference measure. Therefore, it is important to assess the subject's affective state prior to the experiment using standardized questionnaires and to probe the subject's anxiety during the experiment. Typical results show significantly higher anxiety ratings during the threat blocks than during the safe blocks; t(18) = 8.85; p <0.001.
Figure 1: Schematic of a Typical Equipment Setup. (A) Use separate computers to administer the task and record physiological signals from the subject. Synchronize events with the psychophysiology monitoring hardware and the shock device via the parallel port of the experiment computer. Relay the physiological signals from the psychophysiology monitoring hardware to the acquisition computer via the Ethernet cable. Deliver the shock to the subject using the shock device, which is controlled by a signal generator and triggered by the task computer. Deliver the white noise to the subject via the sound card of the task computer and record the trace using the psychophysiology monitoring hardware. (B) Required settings for the signal generator. (C) Required settings for the shock device. Please click here to view a larger version of this figure.
Figure 2: Schematic of a Typical Subject Setup. Attach electrodes to deliver the shock to the subject's non-dominant wrist. Attach electrodes to measure skin conductance on the subject's non-dominant palm. Attach electrodes to measure electromyography below the right eye, over the orbicularis oculi muscle. Attach electrodes to measure electrocardiography on the subject's left bicep and right clavicle. Please click here to view a larger version of this figure.
Figure 3: Schematic of Typical Experimental Design. Present the subjects with a series of letters followed by a brief maintenance period and a response prompt. During the response prompt, present the subjects with a letter (from the series) and a number. Instruct the subjects to indicate whether the number matches the position of the target letter in the previous series. Present startle probes during each trial, either during the maintenance period or the intertrial interval (ITI). Please click here to view a larger version of this figure.
Figure 4: Example EMG Traces Following a White Noise Probe. (A) Raw EMG trace. (B) EMG trace bandpass filtered at 30 to 500 Hz. (C) EMG trace that has been both filtered and rectified using a 20 ms constant. (D) Raw EMG trace from a trial contaminated by baseline noise. Please click here to view a larger version of this figure.
Figure 5: Typical Reaction Time (RT) Results. Subjects are typically faster during low-load trials than during high-load trials. Subjects are also typically faster under threat of shock. Bars represent the mean ± SEM. Please click here to view a larger version of this figure.
Figure 6: Typical Anxiety-potentiated Startle (APS) Results. When startle is probed during the maintenance period (MNT), subjects typically show larger startle potentiation on low-load compared to high-load trials. However, this effect does not hold when startle is probed during the ITI. Bars represent the mean ±SEM. Please click here to view a larger version of this figure.
Figure 7: Typical Accuracy (percent (%) correct) Results. Subjects are typically more accurate during low load trials than high load trials; however, performance does not tend to vary as a function of threat of shock. Bars represent the mean ± SEM. Please click here to view a larger version of this figure.
Supplemental Code Files: Wav file for white noise presentation (40ms_wn.wav.) Please click here to download this file. Code necessary to setup hardware parameters for experimental software (Sternberg_threat_v5.exp.) Please click here to download this file. Code necessary to run experiment (Sternberg_threat_v5.sce.). Please click here to download this file.
This paper demonstrates how to administer the Sternberg WM task during threat of shock. Using this protocol, it was possible to show that WM maintenance is sufficient to reduce anxiety, as measured by the potentiation of the acoustic startle reflex3. These results suggest that the relationship between cognition and anxiety is bidirectional3–5 and that models of anxiety (e.g., the attentional control theory)1 must explain the effect of cognition on anxiety in addition to the effect of anxiety on cognition. Although the current protocol describes the integration of the Sternberg WM task and the threat of shock paradigm, it can also serve as a framework for studying the relationship between cognition and anxiety more generally21.
By redesigning existing cognitive tasks to take place during alternating periods of safety and threat, it is possible to study the effect of anxiety on specific cognitive processes, like WM and sustained attention2,31,32. For instance, in previous work, the N-back working memory task was integrated with the threat of shock paradigm, demonstrating that anxiety interferes with WM at a low load but not a high load4,5. These results suggest that anxiety interferes with WM, but also that healthy individuals are capable of overcoming anxiety when task demands are high. The Sustained Attention to Response Task (SART) was also integrated with the threat of shock paradigm; subjects had to inhibit their responses to infrequent target stimuli. This demonstrated that the threat of shock increases the accuracy on NoGo trials during the task31,32. Together with the N-back studies, these results suggest that anxiety can both impair and facilitate performance, and that the direction of the effect depends on the specific cognitive processes engaged by the task.
Likewise, by adding precisely timed startle probes to an existing cognitive task that has been adapted to the threat of shock paradigm, it is possible to study the effect of specific cognitive tasks on anxiety. The relationship between the WM load and anxiety was initially observed during the N-back WM tasks, where increasing the number of to-be-maintained items reduced APS4,5. However, because this task requires both maintenance and manipulation, it was difficult to determine which WM components were necessary for the observed reduction in anxiety23,33. By following up these studies with the simpler Sternberg WM paradigm, it was possible to show that central executive processing was not necessary for anxiety reduction3.
This technique can be used to study both the effect of anxiety on cognition, as well as the effect of cognition on anxiety. Accordingly, it is important to manipulate both anxiety and cognitive load in this paradigm and to take reliable measures of each. When applying this method to novel cognitive paradigms, it is important to ensure that the cognitive paradigm has distinguishable levels of difficulty based on performance. If pilot testing does not show differences in performance across experimental conditions, check for ceiling/floor effects and adjust the difficulty of the task accordingly. Likewise, it is important to design the threat of shock manipulation such that it is possible to observe APS during conditions of low cognitive load. If pilot testing does not show differences in startle during conditions of low cognitive load, try checking the signal-to-noise ratio in the EMG channel.
There are 3 critical steps to ensuring the effectiveness of this protocol. First, it is important to ensure that the subject understands the cognitive task being implemented. If necessary, design a practice version of the task to ensure that the subjects understand the instructions. Second, it is important to ensure that the electrical stimulation used is of a sufficient intensity to induce anxiety in the subject. If necessary, recalibrate the intensity of the electrical stimulation after each run. Third, it is important to ensure that the signal-to-noise ratio of the EMG channel is sufficient to recover the acoustic startle response. If the channel is noisy or the impedance is too high, thoroughly clean the skin under the eye and reapply the EMG electrodes.
Although there are a number of strengths to this paradigm, there are also limitations that should be addressed. For instance, the use of aversive electrical shock can raise concern among some IRBs, especially when dealing with vulnerable populations. It should be noted that there are alternative approaches to induce anxiety besides using electric shock. These include breathing elevated levels of CO2 (7.5%) for extended periods (8-20 min)34, using the threat of an aversive thermal stimulus35, presenting negatively valenced pictures36, etc. However, it should be noted that electrical stimuli are safe (when used properly), widely used, and effective. Although this protocol recommends a standardization approach to analyze potentiated startle, raw scores may be more reliable in some cases9,10. If standardized scores are used, it is recommended to examine the raw scores as well.
The strength of this protocol is that it allows the researcher to flexibly manipulate state anxiety within-subject in a single session and to test the relationship between anxiety and specific cognitive processes. There are three potential future applications of this protocol. First, it is important to understand how cognitive and emotional systems interact at the level of neural processes. Future studies should examine the relationship between anxiety and WM maintenance-related neural activity, using this paradigm while recording BOLD activity. Second, it is important to generalize these findings to other cognitive processes, such as sustained attention and reward processing. Future studies using this protocol should manipulate these processes during periods of threat and safety. Third, it is important to understand the relationship between cognition and anxiety, in both healthy individuals and in patient populations. Future studies using this protocol should include individuals from these special populations.
In conclusion, this work presents a protocol for studying the relationship between WM load and induced anxiety. Studies using this paradigm have shown that WM maintenance is sufficient to reduce anxiety but that anxiety does not interfere with the WM load itself. Although the findings presented here are specific to the Sternberg WM paradigm, this protocol can be adapted to study the bidirectional relationship between cognition and anxiety in general.
The authors have nothing to disclose.
Financial support for this study was provided by the Intramural Research Program of the National Institute of Mental Health, ZIAMH002798 (ClinicalTrial.gov Identifier: NCT00026559: Protocol ID 01-M-0185).
Biopac System | |||
System | Biopac Systems Inc. | MP150 | 1, Psychophysiology monitoring hardware |
TTL integration | Biopac Systems Inc. | STP100C | 1 |
EDA | Biopac Systems Inc. | EDA100C | 1 |
ECG | Biopac Systems Inc. | ECG100C | 1 |
EMG | Biopac Systems Inc. | EMG100C | 1 |
Name | Company | Catalog Number | Comments |
Other Equipment | |||
Breakout box | See Alternatives | Custom | 1 |
Grass Signal Generator | Grass Instruments | SD9 | 1 |
Shock device | Digitimer North America, LLC | DS7A | 1 |
Name | Company | Catalog Number | Comments |
Alternatives | |||
Alternative to Breakout box | Cortech Solutions | SD-MS-TCPBNC | 1 |
Alternative Grass Signal Generator | Digitimer North America, LLC | DG2A | 1 |
Name | Company | Catalog Number | Comments |
Audio Equipment | |||
Headphones | Sennheiser Electronic GMBH & CO | HD-280 | 1 |
Headphone Amplifier | Applied Research and Technology | AMP4 | 1 |
Sound Pressure Level Meter | Hisgadget Inc | MS10 | 1 |
Name | Company | Catalog Number | Comments |
Electrodes and Leads from Biopac | |||
EMG | Biopac Systems Inc. | EL254S | 2 |
EMG stickers | Biopac Systems Inc. | ADD204 | 2 |
Gel for EMG | Biopac Systems Inc. | GEL100 | 1 |
ECG | Biopac Systems Inc. | LEAD110 | 2 |
Shock | Biopac Systems Inc. | LEAD110 | 2 |
ECG | Biopac Systems Inc. | LEAD110S-W | 1 |
ECG | Biopac Systems Inc. | LEAD110S-R | 1 |
Disposable electrodes | Biopac Systems Inc. | EL508 | 6 |
Name | Company | Catalog Number | Comments |
Software | |||
Presentation | Neurobehavioral Systems | Version 18 | Referred to here as experimental software |
Acknowledge | Biopac Systems Inc. | Version 4.2 | Referred to here as psychophysiology analysis software |