The study was supported by the small fund project of HKU and NSFC.61841704.

This ERP study was approved by The University of Hong Kong Institutional Review Board. Before participating in this study, all participants signed a written informed consent form.
1. Experimental design
<ol>
	<li>Recruit participants
	<ol>
		<li>Recruit participants with at least 1 year (~ 200-3,000 h) of experience in chanting the name of &#34;Amitabha Buddha&#34; for this study. 
		NOTE: In the present study, 21 human participants ranging from 40-52 years old were selected; 11 were males.</li>
	</ol>
	</li>
	<li>Religious chanting vs. nonreligious chanting
	<ol>
		<li>Chant the name of &#34;Amitabha Buddha&#34; for 40 s. First 20 s with the image of Amitabha Buddha and the next 20 s with IAPS images.
		<ol>
			<li>Chant only four characters of the name of &#34;Amitabha Buddha&#34; while viewing the image of the Amitabha Buddha in Pureland school14.</li>
		</ol>
		</li>
		<li>Chant the name of Santa Claus (non-religious chanting condition) for 40 s. First 20 s while viewing the image of the&#160;Santa Claus and the next 20 s with IAPS images.
		<ol>
			<li>Chant only four characters of the name of Santa Claus and imagine the Santa Claus.</li>
		</ol>
		</li>
		<li>Keep silent for 40 s. First 20 s with&#160;a blank image for control purpose and the next 20 s with IAPS images. 
		NOTE: No chanting.</li>
	</ol>
	</li>
	<li>EEG recording system
	<ol>
		<li>Record EEG data using a 128-channel EEG system that consists of an amplifier, headbox, EEG cap, and two desktop computers (see Table of Materials).</li>
	</ol>
	</li>
	<li>Stimuli presenting system
	<ol>
		<li>Use stimulus presentation software (see Table of Materials) to show neutral and negative pictures from the IAPS&#160;on a desktop computer.</li>
	</ol>
	</li>
	<li>ECG recording system
	<ol>
		<li>Use a physiological data recording system to record ECG data (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Affective modulation experiment
NOTE: The experiment had two factors with a 2 x 3 design: The first factor was the picture type: neutral and negative (fear- and stress- provoking). The second factor was the chanting type: chanting &#34;Amitabha Buddha&#34;, chanting &#34;Santa Claus&#34; and no chanting (silent view).
<ol>
	<li>Use a block design, as it may more effectively elicit emotion-related components15. 
	NOTE: There were six conditions, and the sequences were randomized and counterbalanced between the participants (Figure 1). The six conditions were as follows: religious chanting while viewing negative pictures (AmiNeg); religious chanting while viewing neutral pictures (AmiNeu); no chanting while viewing negative pictures (PasNeg); no chanting while viewing neutral pictures (PasNeu); nonreligious chanting while viewing negative pictures (SanNeg); and nonreligious chanting while viewing neutral pictures (SanNeu).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62960/62960fig01.jpg" /> 
Figure 1: The experimental procedure. There were six pseudorandomized conditions, and each participant received a pseudorandomized sequence. Each condition was repeated six times in two separate sessions. This figure has been adapted from Reference3. <a href="https://www.jove.com/files/ftp_upload/62960/62960fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Show each picture for ~1.8-2.2 s, with an interstimulus interval (ISI) of 0.4-0.6 s. 
	NOTE: There were 10 pictures of the same type (neutral or negative) in each session.</li>
	<li>Allow a rest period of 20 s after each session to counter the potential residual effects of chanting or picture viewing on the next session.</li>
	<li>Present the pictures on a CRT monitor at a distance of 75 cm from the participants&#39; eyes, with visual angles of 15&#176; (vertical) and 21&#176; (horizontal).</li>
	<li>Ask the participants to observe the pictures carefully.</li>
	<li>Provide a brief practice run to the participants to allow them to familiarize themselves with each condition. Use a video monitor to ensure that the participants do not fall asleep.</li>
	<li>Give the participants a 10 min rest in the middle of the 40 min experiment.</li>
</ol>
3. EEG and ECG data collection
NOTE: Before coming to the experiment, ask each participant to wash their hair and scalp thoroughly without using a conditioner or anything else that may increase the system&#39;s impedance. Collect the EEG and ECG data simultaneously by two separate systems.
<ol>
	<li>Inform each participant of the experimental procedures, that is, that effective pictures were viewed under different chanting conditions.</li>
	<li>Set the sampling rate to 1,000 Hz, and maintain the impedance of each electrode below 30 k&#937; whenever possible or according to the system&#39;s requirements.</li>
	<li>Collect physiological data, including ECG data using a physiological data recording system (see Table of Materials).</li>
</ol>
4. EEG data analysis
<ol>
	<li>Process and analyze the EEG data with EEGLAB (see Table of Materials) , Supplementary File 1-2, an open-source software16&#160;following the steps below.</li>
	<li>Use the EEGLAB function &#34;pop_resample&#34; to resample the data from 1,000 Hz to 250 Hz to maintain a reasonable data file size. Click on Tools &#62; Change sampling rate.</li>
	<li>Use the EEGLAB function &#34;pop_eegfiltnew&#34; to filter the data with a finite impulse response (FIR) filter with 0.1-100 Hz passband. Click on Tools &#62; Filter the data &#62; Basic FIR filter (new, default).</li>
	<li>Filter the data again with a nonlinear infinite impulse response (IIR) filter with a 47-53 Hz stopband to reduce the noise from the alternating current. Click on Tools &#62; Filter the data &#62; select Notch filter the data instead of pass band.</li>
	<li>Visually inspect the data to remove strong artifacts generated by eye and muscle movements. Click on Plot &#62; Channel data (scroll).</li>
	<li>Visually inspect the data again for any consistent noise generated by any channel, and the bad channels were noted.</li>
	<li>Reconstruct the bad channels using spherical interpolation. Click on Tools &#62; Interpolate electrodes &#62; Select from the data channels.</li>
	<li>Run independent component analysis (ICA) with the open-source algorithm &#34;runica&#34;16. Click on Tools &#62; Run ICA.</li>
	<li>Remove the independent components (ICs) corresponding to eye movements, blinks, muscle movement, and line noise. Click on Tools &#62; Reject data using ICA &#62; Reject components by map.</li>
	<li>Reconstruct the data using the remaining ICs. Click on Tools &#62; Remove components.</li>
	<li>Filter the data with a 30 Hz low-pass filter. Click on Tools &#62; Filter the data &#62; Basic FIR filter (new, default).</li>
	<li>Obtain ERP data by extracting and averaging time-locked epochs for each condition with a time window of -200 to 0 ms as the baseline and 0 to 800 ms as the ERP. Click on Tools &#62; Extract epochs.</li>
	<li>Re-reference the ERP data with the average of the left and right mastoid channels. Click on Tools &#62; Re-reference.</li>
	<li>Repeat the above steps for the datasets from all participants and compare the differences between conditions using the t-test or repeated-measures ANOVA in a statistical analysis software (see Table of Materials).</li>
	<li>Define time windows for N1 and LPP based on established theories8,17and the current data3. 
	NOTE: In this work, N1 was defined as 100-150 ms, while LPP as 300-600 ms from stimulus onset; LPP is most prominent in the central-parietal region (Figure 2).</li>
	<li>Find the neutral vs. negative picture difference at the N1 component using paired t-test among three conditions (Figure 3).</li>
	<li>Find the neutral vs. negative picture difference at the LPP component using paired t-test among three conditions (Figure 4).</li>
	<li>Perform region of interest (ROI) analysis on N1 and LPP components by averaging relevant channels to represent a region. 
	NOTE: To select ROI, the epochs of all three conditions were averaged to calculate those channels where the neutral and negative pictures had a difference that was significant in the specific time window (e.g., for N1 or LPP).</li>
	<li>Compare the difference at N1 and LPP separately, using repeated measures ANOVA and post hoc statistics in statistical analysis software. 
	NOTE: Use post hoc analysis (Bonferroni correction) and determine significant differences between the two conditions separately if the model was significant. The significance threshold was set at p &#60; 0.05.</li>
</ol>
5. ERP source analysis
<ol>
	<li>Perform the ERP source analysis18&#160;with the SPM19&#160;open-source software (see Table of Materials) following the steps below.</li>
	<li>Link the EEG cap sensor coordinate system to the coordinate system of a standard structural MRI image (Montreal Neurological Institute (MNI) coordinates) by landmark-based co-registration. In SPM, click on Batch &#62; SPM &#62; M/EEG &#62; Source reconstruction &#62; Head model specification.</li>
	<li>Perform forward computation to calculate the effect of each dipole on the cortical mesh imposed on the EEG sensors. Under the same Batch Editor, click on SPM &#62; M/EEG &#62; Source reconstruction &#62; Source inversion. 
	NOTE: These results were placed in a G matrix (n x m), where n is the number of sensors (EEG space dimension) and m is the number of mesh vertices (source space dimension). The source model was X = GS, where X is an n x k matrix denoting the ERP data of each condition, k is the number of time points, and S is an m x k matrix indicating the ERP source.</li>
	<li>Use the greedy search-based multiple sparse priors algorithm (since S is unknown) in the third step (among the many algorithms available) to perform the inverse reconstruction because it is more reliable than other methods20. Choose MSP (GS) for the Inversion type in the Source Inversion window.</li>
	<li>Determine the difference between conditions using general linear modeling in SPM. Set the significance level to p &#60; 0.05. Under Batch Editor, click on SPM &#62; Stats &#62; Factorial design specification.</li>
</ol>
6. ECG data and behavioral assessment analysis
<ol>
	<li>Use physiological and data processing software to process and analyze the ECG data (see Table of Materials). Calculate the mean scores for each condition. In EEGLAB, click on Tools &#62; FMRIB Tools &#62; Detect QRS events21. 
	NOTE: Similar to the ERP amplitude analysis, statistical software was used to further analyze the data with repeated measures ANOVA. Post hoc analysis was performed to determine the significant differences between the two conditions separately if the model was significant. The significance level was set to p &#60; 0.05.</li>
	<li>Ask the participants to rate their belief in the efficacy of chanting the subject&#39;s name (Amitabha Buddha, Santa Claus, etc.) on a 1-9 scale, where 1 is considered weakest and 9 the strongest.</li>
</ol>

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The authors declare that they have no competing financial interests.

The uniqueness of this study is the application of a neuroscientific method to probe the neural mechanisms underlying a widespread religious practice, i.e., repetitive religious chanting. Given its prominent effect, this method could enable new interventions for therapists or clinicians to treat clients dealing with emotional problems and suffering from anxiety and stress. Together with previous studies, broader emotion regulation research should be considered in future studies7,8,9,22.
There are few ERP studies on chanting, given the difficulty of constructing experiments that combine chanting and other cognitive events. This study demonstrates a feasible protocol for investigating the affective effect of chanting/praying, which is rather popular in the real world. Previous functional MRI (fMRI) studies found that praying recruits areas of social cognition23. One resting-state fMRI study revealed that chanting &#34;OM&#34; reduced outputs from the anterior cingulate, insula, and orbitofrontal cortices24. Another EEG study found that &#34;OM&#34; meditation increased delta waves, inducing the experience of relaxation and deep sleep25. However, these methods could not precisely investigate the specific event-related changes after religious chanting.
Researchers should control the confounding factors of language processing and familiarity to successfully investigate the potential effect of repetitive religious chanting successfully. As the participants practiced extensively and daily chanting the name &#34;Amitabha Buddha&#34; (Chinese characters: <img alt="Equation 1" src="/files/ftp_upload/62960/62960eq01.jpg" />;&#160;Cantonese pronunciation: o1-nei4-to4-fat6), we used the name &#34;Santa Claus&#34; (Chinese characters: <img alt="Equation 2" src="/files/ftp_upload/62960/62960eq02.jpg" />; Cantonese pronunciation: sing3-daan3-lou5-jan4) as the control condition because the local is familiar with Santa Claus. In Chinese, both names contain four characters, thus controlling for language similarity. Regarding familiarity, Santa Claus is also quite popular in Hong Kong because it is a partially Westernized city. In addition, Santa Claus is also a somewhat positive figure in Hong Kong, where there is official Christmas Holidays. Nevertheless, this control of familiarity is partial, as it is difficult to entirely match the understanding of Amitabha Buddha&#39;s name for the practitioners.
One critical step in the current study was the preparation of the fear- or stress-provoking pictures. As religious chanting may work better when threatening events occur, selecting proper stimuli from the IAPS image pool26&#160;was crucial. It is recommended that potential participants be interviewed and that suitable pictures be chosen to avoid too much fear or disgust. Highly negative pictures could prevent the participants from willfully averting their attention; at the same time, the fear- and stress- provoking stimuli should enable the participants to experience a sufficient threat. Another critical issue is the block design of the study. The EEG/ERP signal is sufficiently sensitive and dynamic to follow every event. However, it would be more appropriate to implement a block design with a 20-30 s viewing period because the pattern of cardiac function or emotion may not change on the order of seconds27. On the other hand, a 60 s block might be too long, and the neural response could become habituated in the ERP studies.
The EEG data processing stage needs to make a backup during each step, as each step alters the data and records the changes made during those steps. This can be used to track changes and make it easier to find errors during batch processing. Improving the data quality is also essential, so experience in raw data cleaning and identifying bad ICs&#160;is needed. In the statistical analysis, comparisons were made on grand averages, and ANOVA was applied. We caution that this statistic with the fixed-effect model is susceptible to random effects28. Mixed-effects models can be adapted to control extraneous factors29, and the assumption of linearity can potentially affect inferences drawn from the ERP data30.
Several limitations are worth noting. One limitation is that the current study enrolled only one group of participants who practiced Pureland Buddhism. Enrolling a control group without any experience in religious chanting for comparison could help determine whether the effect of religious chanting is mediated by belief or familiarity. Usually, a randomized controlled trial would be more convincing to examine the impact of emotion modulation on religious chanting31. However, it is difficult to guarantee that any participant would repetitively chant &#34;Amitabha Buddha&#34; with complete willingness. Additionally, the LPP is affected by other factors, such as emotional sound or positive priming32,33. Thus, better-controlled experiments are needed to delineate more clearly the fundamental neuro-mechanism underlying the effect of religious chanting.
In sum, previous studies have demonstrated that the human brain is subjective to neural plasticity and swift alteration of states34,35; with sufficient practice and intention, the brain can reshape itself and respond differently to normally fearful stimuli. This study provides insights into the development of effective coping strategies for handling emotional distress in contemporary contexts. Following this protocol, researchers should examine the effect of religious chanting or other traditional practices to identify feasible ways to help people ameliorate their emotional sufferings.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62960">Modulation of the Neurophysiological Response to Fearful and Stressful Stimuli Through Repetitive Religious Chanting</a>. The Authors section was&#160;updated.
The authors section was updated from:
Hin Hung Sik1, Georgios T. Halkias1, Chunqi Chang2, Junling Gao1, Hang Kin Leung1, Bonnie W. Y. Wu1 
1Centre of Buddhist Studies, The University of Hong Kong, 
2School of Biomedical Engineering, Shenzhen University
To:
Hin Hung Sik1, Georgios T. Halkias1, Chunqi Chang2, Junling Gao1, Hang Kin Leung1, Bonnie Wai&#160;Yan&#160;Wu1 
1Buddhism and Science Research Lab, Centre of Buddhist Studies, The University of Hong Kong, 
2School of Biomedical Engineering, Shenzhen University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62960">Modulation of the Neurophysiological Response to Fearful and Stressful Stimuli Through Repetitive Religious Chanting</a>. The Authors section was&#160;updated.

Erratum: Modulation of the Neurophysiological Response to Fearful and Stressful Stimuli Through Repetitive Religious Chanting

The late positive potential (LPP) has long been accompanied by emotional arousal, and it has been reliably used in emotion-related research1,2. Religious practice is widespread in both Eastern and Western countries. It is asserted that it can alleviate the practitioner&#39;s anxiety and stress when facing adverse events, especially during times of difficulty3. Nonetheless, this has seldom been demonstrated under rigorous experimental settings.
Numerous studies have confirmed that emotion regulation can be learned with different strategies and frameworks4,5,6. A few studies have shown that mindfulness and meditation can modulate the neural response to affective events7,8. Recently, it was found that meditation practitioners may employ emotion modulation strategies other than cognitive appraisal, suppression, and distraction8,9. Stimuli from the International Affective Picture System (IAPS) can be used to elicit positive or negative emotions reliably, and there are standard criteria for finding designed pictures with specified valence and arousal levels in affective research10.
Emotional stimuli can cause early- and later- responses in the brain3,11. Similarly, Buddhism tradition made analogical analysis on the mind thoughts by initial and secondary mental processes3,12,13. The Sallatha Sutta (The Arrow Sutta), an early Buddhist text, mentions that cognitive training can tame emotion. The Arrow Sutta states that both a well-trained Buddhist practitioner and an untrained person experience an initial and negative perception of pain when facing a harmful event13. This unavoidable initial pain is similar to a person being hit by an arrow, as described in the Sallatha Sutta. Early perceptual pain is identical to the stage of early processing when a person views a highly negative picture. Early neural processing usually elicits an N1 component. Untrained persons may develop excessive emotions, such as worry, anxiety, and stress, after experiencing the initial, unavoidable painful feelings. According to the Sallatha Sutta, this late-developing negative emotion or psychological pain is like being hit by a second arrow. An event-related potential (ERP) experiment may capture the current design&#39;s early and later psychological processes, assuming that N1 and LPP could correspond to the two arrows mentioned above.
In this protocol, the repetitive chanting of the name &#34;Amitabha Buddha&#34; (Sanskit: Amit&#257;bha) was chosen to test the potential effect of religious chanting when an individual is in a fearful or stressful situation. This religious chanting is one of the most popular practices of individuals with religious orientations among Chinese Buddhists, and it is a core practice of East Asian Pure Land Buddhism14. It was hypothesized that repetitive religious chanting would reduce the brain response to provoking stimuli, namely, the LPP induced by fearful or stressful pictures. Both EEG and electrocardiogram (ECG) data were collected to assess participants&#39; neurophysiological responses under different conditions.

<table><tbody><tr><td>E-Prime 2.0</td><td>Psychology Software Tools</td><td></td><td>stimulus presentation, behavior data collection</td></tr><tr><td>EEGLAB</td><td>Swartz Center for Computational Neuroscience</td><td></td><td>EEG analysis software</td></tr><tr><td>HydroCel GSN 128 channels</td><td>Electrical Geodesics, Inc. (EGI)</td><td></td><td>EEG cap</td></tr><tr><td>LabChart</td><td>ADInstruments</td><td></td><td>physiological data (including ECG) acquisition software</td></tr><tr><td>Matlab R2011a</td><td>MathWorks</td><td></td><td>EEGLAB and SPM are based on Matlab; statistical analysis tool for EEG and physiological data</td></tr><tr><td>Netstation</td><td>Electrical Geodesics, Inc. (EGI)</td><td></td><td>EEG acquisition software</td></tr><tr><td>PowerLab 8/35</td><td>ADInstruments</td><td>PL3508</td><td>physiological data (including ECG) acquisition hardware</td></tr><tr><td>SPM</td><td>Wellcome Trust Centre for Neuroimaging</td><td></td><td>EEG source analysis software</td></tr><tr><td>FMRIB</td><td>University of Oxford Centre for Functional MRI of the Brain (FMRIB)</td><td></td><td>Plug-in for EEGLAB to process ECG data</td></tr><tr><td>SPSS</td><td>IBM</td><td></td><td>statistical analysis tool for behavior and EEG ROI data</td></tr><tr><td>iMac 27&quot;</td><td>Apple</td><td></td><td>running the Netstation software</td></tr><tr><td>Windows PC</td><td>HP</td><td></td><td>running the E-Prime 2.0 software</td></tr><tr><td>Windows PC</td><td>Dell</td><td></td><td>running the LabChart software</td></tr></tbody></table>

modulation of the neurophysiological response to fearful and stressful stimuli through repetitive religious chanting

In neuropsychological experiments, the late positive potential (LPP) is an event-related potential (ERP) component that reflects the level of one's emotional arousal. This study investigates whether repetitive religious chanting modulates the emotional response to fear- and stress-provoking stimuli, thus leading to a less responsive LPP. Twenty-one participants with at least one year of experience in the repetitive religious chanting of "Amitabha Buddha" were recruited. A 128-channel electroencephalography (EEG) system was used to collect EEG data. The participants were instructed to view negative or neutral pictures selected from the International Affective Picture System (IAPS) under three conditions: repetitive religious chanting, repetitive nonreligious chanting, and no chanting. The results demonstrated that viewing the negative fear- and stress-provoking pictures induced larger LPPs in the participants than viewing neutral pictures under the no-chanting and nonreligious chanting conditions. However, this increased LPP largely disappeared under repetitive religious chanting conditions. The findings indicate that repetitive religious chanting may effectively alleviate the neurophysiological response to fearful or stressful situations for practitioners.

Behavioral results 
The results for participants&#39; belief of chanting revealed an average score of 8.16 &#177; 0.96 for &#34;Amitabha Buddha&#34;, 3.26 &#177; 2.56 for &#34;Santa Claus&#34;, and 1.95 &#177; 2.09 for the blank control condition (Supplementary Table 1).
ERP results 
The representative channel of Pz (parietal lobe) demonstrated that the chanting conditions had different effects on the early (N1) and late (LPP) processing of neutral and negative pictures. It showed the time window of N1 and LPP, respectively (Figure 2).
Early perceptual stage 
The&#160;ERP results showed an increased N1 while viewing the negative pictures in three chanting conditions (Figure 3). It showed that negative images induced stronger central brain activities than neutral images, and the increases are comparable in three conditions.
Late emotional/cognitive stage 
The ERP demonstrated an increased LPP in the nonreligious chanting and no-chanting conditions. However, the LPP induced by negative pictures is barely visible when the participant chants Amitabha Buddha&#39;s name (Figure 4).
Region of interest (ROI) analysis 
The three conditions were combined to estimate the regions that were generally activated at N1 and LPP components. Repeated measures ANOVA was performed with statistical software to calculate the difference in the N1 and LPP components between the chanting conditions (Figure 5).
The left three columns show the difference in the N1 component for the three chanting conditions: the silent viewing condition, the nonreligious chanting condition, and the religious chanting condition. The differences in the N1 component were similar across the three conditions. The right three columns show the difference in the LPP component for the three chanting conditions. This demonstrates that the difference in the LPP component is much smaller in the religious chanting condition than in the nonreligious chanting condition and the silent viewing condition.
Source analysis 
Source analysis was applied to extract the potential brain mapping based on the LPP results (Figure 6). The results show that when compared with neutral pictures, negative pictures induce more parietal activation in the nonreligious chanting condition and no chanting condition. In contrast, this negative picture-induced activation largely disappears in the religious chanting condition.
Physiological results: heart rate 
There was a significant change in the heart rate (HR) between the negative and neutral pictures in the nonreligious chanting condition. A similar trend was found in the no-chanting condition. However, no such HR difference was found in the religious chanting condition (Figure 7).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62960/62960fig02.jpg" /> 
Figure 2: A representative channel (Pz) showed different ERPs in six chanting conditions. The six conditions are (1) religious chanting while viewing neutral pictures (AmiNeu); (2) religious chanting while viewing negative pictures (AmiNeg); (3) nonreligious chanting while viewing neutral pictures (SanNeu); (4) nonreligious chanting while viewing negative pictures (SanNeg); (5) no chanting while viewing neutral pictures (PasNeu); and (6) no chanting while viewing negative pictures (PasNeg). The channel Pz located in the mid-parietal area of the scalp.&#160;<a href="https://www.jove.com/files/ftp_upload/62960/62960fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62960/62960fig3v2.jpg" /> 
Figure 3: The ERP results for demonstrating the N1 component in the three chanting conditions. Two-dimensional maps of the N1 component for the three conditions for each picture type. In the last column, channels with significant differences (p &#60; 0.05) are shown with dots; dots that are in darker color indicate greater significance (i.e., smaller p-values). <a href="https://www.jove.com/files/ftp_upload/62960/62960fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62960/62960fig4v2.jpg" /> 
Figure 4: The ERP results for demonstrating the LPP component in the three chanting conditions. Two-dimensional maps of the late positive potential (LPP) component for the three conditions for each picture type. In the last column, channels with significant differences (p &#60; 0.05) are shown with dots; dots that are in darker color indicate greater significance (i.e., smaller p-values). <a href="https://www.jove.com/files/ftp_upload/62960/62960fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62960/62960fig5v2.jpg" /> 
Figure 5: Region of interest (ROI) analysis. The&#160;Region of interest (ROI) analysis on the difference between negative vs. neutral picture-induced brain responses for the early component, N1, and the late component, the late positive potential (LPP). <a href="https://www.jove.com/files/ftp_upload/62960/62960fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62960/62960fig06.jpg" /> 
Figure 6: Source analysis of the late positive potential (LPP) component under the three conditions. Highlighted areas indicates higher brain activity in negative vs. neutral conditions.&#160;<a href="https://www.jove.com/files/ftp_upload/62960/62960fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62960/62960fig7v2.jpg" /> 
Figure 7: The heart beat intervals under the three chanting conditions. The electrocardiogram&#39;s inter-beat intervals (RRs) under each picture type/chanting combination and the corresponding p values. Ami: Amitabha Buddha chanting condition, San: Santa Claus chanting condition, Pas: passive viewing condition, Neu: neutral picture, Neg: negative picture. <a href="https://www.jove.com/files/ftp_upload/62960/62960fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Rating the belief in the efficacy of the chanting subject (Amitabha Buddha, Santa Claus). It uses a 1-9 scale, where 1 indicates the least belief and 9 the strongest belief. <a href="https://www.jove.com/files/ftp_upload/62960/62960_Supplementary Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Code for EEG data batch preprocessing. It removes bad channels, resamples the data to 250 Hz, and then filters the data. <a href="https://www.jove.com/files/ftp_upload/62960/EEG_Preprocess.m" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Code for ERP data repairing. It repairs bad epochs with noisy spikes. <a href="https://www.jove.com/files/ftp_upload/62960/ERP_Repair.m" target="_blank">Please click here to download this File.</a>

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Modulation of the Neurophysiological Response to Fearful and Stressful Stimuli Through Repetitive Religious Chanting

The present event-related potential (ERP) study provides a unique protocol for investigating how religious chanting can modulate negative emotions. The results demonstrate that the late positive potential (LPP) is a robust neurophysiological response to negative emotional stimuli and can be effectively modulated by repetitive religious chanting.

In neuropsychological experiments, the late positive potential (LPP) is an event-related potential (ERP) component that reflects the level of one's ...

modulation-neurophysiological-response-to-fearful-stressful-stimuli

Research

JoVE Journal

Neuroscience

3.4K Views.  The University of Hong Kong. The present event-related potential (ERP) study provides a unique protocol for investigating how religious chanting can modulate negative emotions. The results demonstrate that the late positive potential (LPP) is a robust neurophysiological response to negative emotional stimuli and can be effectively modulated by repetitive religious chanting.

Video: Modulation of the Neurophysiological Response to Fearful and Stressful Stimuli Through Repetitive Religious Chanting

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.

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 ...

Behavior

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

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 ...

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 ...

A Community-based Stress Management Program: Using Wearable Devices to Assess Whole Body Physiological Responses in Non-laboratory Settings

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches for emotional regulation and optimizing compensatory reserve mechanisms. This protocol is designed to not only teach mind-body awareness but also to provide feedback utilizing physiological data and survey results. The primary findings of this study showed that heart coherence and alpha variables were significantly correlated after four weeks of the breath-based meditation stress protocol. Meditation and rhythmic breathing produced significant increases in alpha brain activity. These brain physiological responses conformed to the Pleth Variability Index (PVI) changes, suggesting the ability of the human body to enter into a meditative state and effectively manage stress. When assessed after four weeks of daily practicing the techniques employed in the stress management protocol, based on the Five Facet Mindfulness Questionnaire, subjects improved in applying mindfulness skills. The overall mindfulness score, Pleth variability index (PVI), and perfusion index (PI) increased after the 4-week intervention period. Results from electroencephalography (brain waves) were consistent with a meditative state during the post-study follow-up session. This provides evidence that wearable devices are feasible for data collection during a breath-based stress management intervention. This protocol can be easily and efficiently implemented into any study design in which physiological data are desired in a non-laboratory-based setting.

Stress is an unavoidable and persistent component of life and holistic approaches for its management are being considered. A standardized methodology was created to demonstrate the feasibility of a breath-based stress management protocol that can be used with commercially available portable technology.

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

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 ...

Decoding Brain Activity During Religious Chanting with EEG and fMRI

Religious Chanting and Self-Related Brain Regions: A Multi-Modal Neuroimaging Study

This protocol presents a multi-modal neuroimaging approach to explore the potential brain activity associated with repetitive religious chanting, a widespread form of mind training in both Eastern and Western cultures. High-density electroencephalogram (EEG), with its superior temporal resolution, allows for capturing the dynamic changes in brain activity during religious chanting. Through source localization methods, these can be attributed to various alternative potential brain region sources. Twenty practitioners of religious chanting were measured with EEG. However, the spatial resolution of EEG is less precise, in comparison to functional magnetic resonance imaging (fMRI). Thus, one highly experienced practitioner underwent an fMRI scanning session to guide the source localization more precisely. The fMRI data helped guide the selection of EEG source localization, making the calculation of K-means of the EEG source localization in the group of 20 intermediate practitioners more precise and reliable. This method enhanced EEG&#39;s ability to identify the brain regions specifically engaged during religious chanting, particularly the cardinal role of the posterior cingulate cortex (PCC). The PCC is a brain area related to focus and self-referential processing. These multimodal neuroimaging and neurophysiological results reveal that repetitive religious chanting can induce lower centrality and higher delta-wave power compared to non-religious chanting and resting state conditions. The combination of fMRI and EEG source analysis provides a more detailed understanding of the brain&#39;s response to repetitive religious chanting. The protocol contributes significantly to the research on the neural mechanisms involved in religious and meditative practices, which is becoming more prominent nowadays. The results of this study could have significant implications for developing future neurofeedback techniques and psychological interventions.

Author Spotlight: Exploring Dynamic Neural Changes Associated with Religious Chanting

Here, we present a protocol to investigate the neurophysiological correlates of various meditation forms, including religious chanting. This method uniquely integrates fMRI eigenvector results for region selection in electroencephalogram (EEG) source analysis using k-means clustering. The results provide an in-depth understanding of the neural processes involved in repetitive religious chanting.

This protocol presents a multi-modal neuroimaging approach to explore the potential brain activity associated with repetitive religious chanting, a ...

Fear Conditioning

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to a fear of the harmless stimulus. This process is largely mediated by the amygdala, which is a brain region involved in emotions and stress reactions. Fear conditioning can be utilized in several ways to understand different aspects of learning and memory.
This video presents an overview of the principles behind fear conditioning, discusses the equipment and a generalized procedure used for this type of experiment. Finally, we'll review some real world applications of fear conditioning in behavioral neuroscience research today.

Fear Conditioning- Classical Conditioning: Principle and Procedure

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to ...

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Psychology

Behavioral Science

Modeling Social Stress

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are unfamiliar with or have difficulty adjusting to. Since it is hard to examine mechanisms of social stress in humans, modeling this condition in animals may help scientist in developing new therapies for treating this commonly encountered problem.
This science education video begins by discussing the known anatomy and physiology behind stress response. Then, we explain a well-established paradigm for modeling social stress in rodents, the Resident-Intruder task. In the applications section, we review some example studies in which response to stress is measured.

Physiology of Stress; Social Stress- Resident-Intruder Task

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are ...