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<journal-id journal-id-type="publisher-id">Front. Neurosci.</journal-id>
<journal-title>Frontiers in Neuroscience</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Neurosci.</abbrev-journal-title>
<issn pub-type="epub">1662-453X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
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<article-meta>
<article-id pub-id-type="doi">10.3389/fnins.2024.1477181</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Neuroscience</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Neuroplastic changes induced by long-term <italic>Pingju</italic> training: insights from dynamic brain activity and connectivity</article-title>
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<name><surname>Zhao</surname> <given-names>Fangshi</given-names></name>
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<name><surname>Song</surname> <given-names>Linlin</given-names></name>
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<name><surname>Chen</surname> <given-names>Yule</given-names></name>
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<name><surname>Wang</surname> <given-names>Shaoying</given-names></name>
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<name><surname>Wang</surname> <given-names>Xiaoyi</given-names></name>
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<name><surname>Zhai</surname> <given-names>Ying</given-names></name>
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<name><surname>Zhang</surname> <given-names>Zhihui</given-names></name>
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<name><surname>Lei</surname> <given-names>Minghuan</given-names></name>
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<name><surname>Zhu</surname> <given-names>Dan</given-names></name>
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<name><surname>Li</surname> <given-names>Fengtan</given-names></name>
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<name><surname>Wang</surname> <given-names>Chunyang</given-names></name>
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<name><surname>Liu</surname> <given-names>Feng</given-names></name>
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<aff id="aff1"><sup>1</sup><institution>Department of Radiology and Tianjin Key Laboratory of Functional Imaging and Tianjin Institute of Radiology, Tianjin Medical University General Hospital</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff>
<aff id="aff2"><sup>2</sup><institution>Department of Ultrasound, Tianjin Medical University General Hospital</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff>
<aff id="aff3"><sup>3</sup><institution>Department of Radiology, The Second Hospital of Tianjin Medical University</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff>
<aff id="aff4"><sup>4</sup><institution>Department of Radiology, Tianjin Medical University General Hospital Airport Hospital</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff>
<aff id="aff5"><sup>5</sup><institution>Department of Scientific Research, Tianjin Medical University General Hospital</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff>
<author-notes>
<fn fn-type="edited-by"><p>Edited by: Minghao Dong, Xidian University, China</p></fn>
<fn fn-type="edited-by"><p>Reviewed by: Yuanchao Zhang, University of Electronic Science and Technology of China, China</p>
<p>Yuanqiang Zhu, Fourth Military Medical University, China</p>
<p>Lijun Bai, Xi&#x2019;an Jiaotong University, China</p></fn>
<corresp id="c001">&#x002A;Correspondence: Feng Liu, <email>fengliu@tmu.edu.cn</email></corresp>
<corresp id="c002">Chunyang Wang, <email>cwang@tmu.edu.cn</email></corresp>
<corresp id="c003">Fengtan Li, <email>left9999@sina.com</email></corresp>
<fn fn-type="equal" id="fn002"><p><sup>&#x2020;</sup>These authors have contributed equally to this work</p></fn>
</author-notes>
<pub-date pub-type="epub">
<day>27</day>
<month>09</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>18</volume>
<elocation-id>1477181</elocation-id>
<history>
<date date-type="received">
<day>07</day>
<month>08</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>09</day>
<month>09</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2024 Zhao, Song, Chen, Wang, Wang, Zhai, Xu, Zhang, Lei, Cai, An, Zhu, Li, Wang and Liu.</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Zhao, Song, Chen, Wang, Wang, Zhai, Xu, Zhang, Lei, Cai, An, Zhu, Li, Wang and Liu</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p></license>
</permissions>
<abstract>
<sec>
<title>Background</title>
<p>Traditional Chinese opera, such as <italic>Pingju</italic>, requires actors to master sophisticated performance skills and cultural knowledge, potentially influencing brain function. This study aimed to explore the effects of long-term opera training on the dynamic amplitude of low-frequency fluctuation (dALFF) and dynamic functional connectivity (dFC).</p>
</sec>
<sec>
<title>Methods</title>
<p>Twenty professional well-trained <italic>Pingju</italic> actors and twenty demographically matched untrained subjects were recruited. Resting-state functional magnetic resonance imaging (fMRI) data were collected to assess dALFF differences in spontaneous regional brain activity between the actors and untrained participants. Brain regions with altered dALFF were selected as the seeds for the subsequent dFC analysis. Statistical comparisons examined differences between groups, while correlation analyses explored the relationships between dALFF and dFC, as well as the associations between these neural measures and the duration of <italic>Pingju</italic> training.</p>
</sec>
<sec>
<title>Results</title>
<p>Compared with untrained subjects, professional <italic>Pingju</italic> actors exhibited significantly lower dALFF in the right lingual gyrus. Additionally, actors showed increased dFC between the right lingual gyrus and the bilateral cerebellum, as well as between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus, compared with untrained subjects. Furthermore, a negative correlation was found between the dALFF in the right lingual gyrus and its dFC, and a significant association was found between dFC in the bilateral midbrain/red nucleus/thalamus and the duration of <italic>Pingju</italic> training.</p>
</sec>
<sec>
<title>Conclusion</title>
<p>Long-term engagement in <italic>Pingju</italic> training induces neuroplastic changes, reflected in altered dALFF and dFC. These findings provide evidence for the interaction between artistic training and brain function, highlighting the need for further research into the impact of professional training on cognitive functions.</p>
</sec>
</abstract>
<kwd-group>
<kwd><italic>Pingju</italic></kwd>
<kwd>neuroplasticity</kwd>
<kwd>dynamic ALFF</kwd>
<kwd>dynamic FC</kwd>
<kwd>resting-state fMRI</kwd>
</kwd-group>
<counts>
<fig-count count="4"/>
<table-count count="2"/>
<equation-count count="0"/>
<ref-count count="62"/>
<page-count count="9"/>
<word-count count="6815"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Neurodevelopment</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="S1" sec-type="intro">
<title>Introduction</title>
<p><italic>Pingju</italic>, also known as Ping Opera, is one of the most popular traditional Chinese operas, particularly prevalent in northern China. It combines a variety of performing arts, including singing, dialogue, acting, and acrobatics, to tell stories that reflect Chinese culture and history (<xref ref-type="bibr" rid="B28">Liu, 2023</xref>). The training for <italic>Pingju</italic> begins at a young age, typically around 6&#x2013;7 years old, and involves rigorous and prolonged practice in vocal techniques, body movements, and emotional expression. Extensive training often leads to significant changes in the brain, a phenomenon known as neuroplasticity, which allows the brain to modify its structure and function in response to new experiences, skills, and environmental changes (<xref ref-type="bibr" rid="B59">Zhang et al., 2018</xref>; <xref ref-type="bibr" rid="B60">Zhao et al., 2020</xref>; <xref ref-type="bibr" rid="B11">Draganski et al., 2004</xref>) and is crucial for learning, memory, and recovery from injury (<xref ref-type="bibr" rid="B50">Wu et al., 2020</xref>; <xref ref-type="bibr" rid="B38">Olszewska et al., 2021</xref>; <xref ref-type="bibr" rid="B20">Hiraga et al., 2022</xref>). For instance, professional musicians and athletes show distinct neural adaptations due to their specialized training (<xref ref-type="bibr" rid="B34">M&#x00FC;nte et al., 2002</xref>; <xref ref-type="bibr" rid="B35">Nager et al., 2020</xref>; <xref ref-type="bibr" rid="B58">Zhang et al., 2021</xref>; <xref ref-type="bibr" rid="B15">Gao et al., 2023</xref>). The unique combination of skills required in <italic>Pingju</italic> provides an opportunity to study the effects of long-term, specialized training on brain function.</p>
<p>To explore the neural mechanisms related to specialized training, researchers can employ resting-state functional magnetic resonance imaging (rs-fMRI), a non-invasive method that captures spontaneous brain activity by detecting fluctuations in blood oxygen level-dependent (BOLD) signals (<xref ref-type="bibr" rid="B16">Guerra-Carrillo et al., 2014</xref>). Previous rs-fMRI studies have consistently shown that specialized training in various professions, such as musicians, athletes, dancers, and seafarers, can result in changes in brain response patterns (<xref ref-type="bibr" rid="B29">Lu et al., 2018</xref>; <xref ref-type="bibr" rid="B48">Wang et al., 2018</xref>; <xref ref-type="bibr" rid="B47">Vuust et al., 2022</xref>; <xref ref-type="bibr" rid="B57">Zhang et al., 2024</xref>). Within rs-fMRI, the amplitude of low-frequency fluctuation (ALFF) and functional connectivity (FC) are two key metrics. ALFF quantifies the power of low-frequency oscillations in the BOLD signal, providing a measure of spontaneous local brain activity (<xref ref-type="bibr" rid="B62">Zuo et al., 2010</xref>; <xref ref-type="bibr" rid="B26">Liu et al., 2013</xref>; <xref ref-type="bibr" rid="B46">Su et al., 2022</xref>). On the other hand, FC measures the temporal correlation between BOLD signals from different brain regions, reflecting the functional interactions and synchronization between these regions (<xref ref-type="bibr" rid="B13">Friston, 2011</xref>; <xref ref-type="bibr" rid="B25">Liu et al., 2015</xref>; <xref ref-type="bibr" rid="B43">Rubia et al., 2019</xref>). Together, ALFF and FC offer a comprehensive view of brain function, linking local neural activity with broader interactions between brain regions. This dual approach allows for a deeper understanding of how specific brain regions and FCs contribute to the skills required in <italic>Pingju</italic> performance.</p>
<p>Traditional static ALFF and FC methods provide an averaged view of brain activity over the entire scanning period, which can miss transient yet important neural dynamics. The brain is a highly dynamic system with constantly changing neural activities and interactions, resulting in time-varying patterns of brain activity (<xref ref-type="bibr" rid="B8">Chang and Glover, 2010</xref>). Relying solely on averaged brain activity across a time series can, therefore, lead to a loss of valuable insights into underlying neuronal processes. To address this issue, dynamic approaches have been developed to capture the temporal variability of brain activities (<xref ref-type="bibr" rid="B21">Hutchison et al., 2013</xref>; <xref ref-type="bibr" rid="B1">Allen et al., 2014</xref>; <xref ref-type="bibr" rid="B27">Liu et al., 2017</xref>). Dynamic fluctuations in resting-state brain activity have been associated with cognitive and behavioral traits, suggesting that dynamic measures may provide more sensitive biomarkers than static ones in certain contexts. For example, dynamic ALFF (dALFF) captures changes in local brain activity over short time frames, highlighting regional neural dynamics associated with processes like learning and adaptation (<xref ref-type="bibr" rid="B61">Zhu et al., 2024</xref>). Similarly, dynamic FC (dFC) monitors shifts in connectivity between brain regions, unveiling changes in network interactions and integration (<xref ref-type="bibr" rid="B42">Preti et al., 2017</xref>), with dFC patterns linked to various cognitive functions and adaptive processes (<xref ref-type="bibr" rid="B51">Xu et al., 2022</xref>; <xref ref-type="bibr" rid="B24">Li et al., 2023</xref>; <xref ref-type="bibr" rid="B33">Matkovic et al., 2023</xref>). Thus, employing a dynamic approach enables the exploration of how brain activity adapts and reorganizes in response to specific demands, such as those encountered in rigorous <italic>Pingju</italic> training, offering a more comprehensive understanding of brain function and its plasticity following long-term training.</p>
<p>In this study, we aimed to explore the neuroplastic effects of <italic>Pingju</italic> training by comparing dynamic brain activity and connectivity between professional <italic>Pingju</italic> actors and untrained individuals. Specifically, we utilized dALFF to measure local brain activity and dFC to assess the functional interactions between brain regions. We hypothesized that the intensive, long-term training of professional <italic>Pingju</italic> actors would lead to different patterns of brain activity and connectivity, reflecting the specialized skills and cognitive demands of their profession.</p>
</sec>
<sec id="S2" sec-type="materials|methods">
<title>Materials and methods</title>
<sec id="S2.SS1">
<title>Participants</title>
<p>This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, and written informed consent was obtained from each participant. The study included 20 professional traditional Chinese <italic>Pingju</italic> actors and 20 untrained individuals. The actors were recruited from Tianjin <italic>Pingju</italic> Theater and Tianjin Baipai <italic>Pingju</italic> Theater. Untrained individuals were those without any professional musical education. None of the participants had a history of neurological or psychiatric disorders, nor did their first-degree relatives. All participants, both actors and untrained subjects, were right-handed according to the criteria of the Chinese revised version of the Edinburgh Handedness Inventory (<xref ref-type="bibr" rid="B37">Oldfield, 1971</xref>).</p>
</sec>
<sec id="S2.SS2">
<title>Image acquisition</title>
<p>MRI data were acquired using a 3.0-Tesla MR system (Discovery MR750, General Electric, Milwaukee, WI, USA). The participants were instructed to stay awake with their eyes closed during the scan. Foam pads were used to minimize head motion, and earplugs were used to reduce scanner noise. The rs-fMRI data were obtained using a single-shot echo-planar sequence with the following parameters: repetition time (TR) = 2000 ms, echo time (TE) = 45 ms, flip angle = 90&#x00B0;, slice thickness = 4.0 mm, gap = 0.5 mm, field of view (FOV) = 220 mm &#x00D7; 220 mm, matrix = 64 &#x00D7; 64, number of axial slices = 32, and 180 volumes. High-resolution T1-weighted images were acquired using a brain volume sequence with TR = 8.17 ms, TE = 3.18 ms, inversion time = 450 ms, slice thickness = 1.0 mm, FOV = 256 mm &#x00D7; 256 mm, matrix = 256 &#x00D7; 256, and 188 sagittal slices.</p>
</sec>
<sec id="S2.SS3">
<title>Image preprocessing</title>
<p>The rs-fMRI data were preprocessed using the Data Processing and Analysis for Brain Imaging (DPABI) toolbox (<xref ref-type="bibr" rid="B53">Yan et al., 2016</xref>). Initially, the first 10 time points were removed to allow signal equilibrium. Subsequently, the fMRI data were corrected for temporal differences between slices and head motion. Participants with a maximum displacement greater than 3.0 mm, maximum rotation greater than 3.0&#x00B0;, or mean framewise displacement (FD) greater than 0.3 mm were excluded from further analyses (<xref ref-type="bibr" rid="B41">Power et al., 2012</xref>). Following this, individual structural images were co-registered to the corresponding mean functional images, and the transformed structural images were segmented into gray matter, white matter, and cerebrospinal fluid. Using these segmented images, normalization parameters from individual native space to Montreal Neurological Institute (MNI) space were estimated via the Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) tool (<xref ref-type="bibr" rid="B2">Ashburner, 2007</xref>). The motion-corrected functional imaging data were then normalized to MNI space using these parameters and resampled to 3-mm cubic voxels. Next, spatial smoothing was conducted with an 8 mm full width at half maximum (FWHM) Gaussian kernel. Finally, spurious variances were removed through regression, including linear drift, Friston-24 head motion parameters, and signals from white matter and cerebrospinal fluid (<xref ref-type="bibr" rid="B30">Ma et al., 2022</xref>).</p>
</sec>
<sec id="S2.SS4">
<title>dALFF calculation</title>
<p>The dALFF analysis was conducted using a sliding-window approach based on the Temporal Dynamic Analysis (TDA) toolkit (<xref ref-type="bibr" rid="B54">Yan et al., 2017</xref>). Since there is no consensus about the selection of window length, a previous study suggests that the minimum window length should not be less than 1/<italic>f</italic><sub><italic>min</italic></sub> (the minimum frequency of the time series), as a shorter window may increase the risk of false fluctuations while a longer window could diminish the dynamics (<xref ref-type="bibr" rid="B22">Leonardi and Van De Ville, 2015</xref>). Based on this guidance, a window length of 50 TRs (100 seconds) and a step size of 1 TR (2 seconds) was used to characterize dALFF (<xref ref-type="bibr" rid="B14">Fu et al., 2018</xref>; <xref ref-type="bibr" rid="B10">Cui et al., 2020</xref>). Within each time window, the ALFF map was calculated by applying a fast Fourier transform (FFT) to convert the time series into the frequency domain, decomposing the signal into its constituent frequencies to obtain the power spectrum (<xref ref-type="bibr" rid="B56">Zang et al., 2007</xref>). The ALFF value was then computed by taking the square root of the power spectrum within the predefined low-frequency range (0.01&#x2013;0.08 Hz). The temporal variability of the ALFF (i.e., dALFF) was assessed by calculating the standard deviation (SD) of these maps. For each participant, the dALFF of each voxel was converted into <italic>z</italic>-scores by subtracting the mean and dividing by the SD of the global values.</p>
</sec>
<sec id="S2.SS5">
<title>dFC analysis</title>
<p>Before dFC analysis, the fMRI data were temporally bandpass-filtered (0.01&#x2013;0.08 Hz) to minimize the effects of low-frequency drift and high-frequency noise. Then, each brain region with altered ALFF was used as a region of interest (ROI) for seed-based dFC analysis (<xref ref-type="bibr" rid="B23">Li et al., 2019</xref>; <xref ref-type="bibr" rid="B9">Christiaen et al., 2020</xref>). The same sliding-window parameters used in the dALFF analysis were applied to derive windowed time series. Within each window, whole-brain FC maps for each ROI were computed by calculating Pearson correlation coefficients between the average time series of all voxels in the ROI and the time series of all other brain voxels. Fisher&#x2019;s <italic>r</italic>-to-<italic>z</italic> transformation was applied to all FC maps to improve normality. The SD of the FC maps across time windows was calculated for each subject to characterize variations in individual ROI-to-whole-brain FC (<xref ref-type="bibr" rid="B17">Harlalka et al., 2019</xref>; <xref ref-type="bibr" rid="B52">Xue et al., 2022</xref>).</p>
</sec>
<sec id="S2.SS6">
<title>Statistical analysis</title>
<p>Voxel-based comparisons of dALFF between the two groups were conducted using a general linear model, with gender and age as covariates. Cluster-level family-wise error (FWE) correction was applied to control for multiple comparisons with a voxel-level uncorrected threshold of <italic>p</italic> &#x003C; 0.001 and a cluster-level corrected threshold of <italic>p</italic> &#x003C; 0.05. The same statistical model and threshold were used for seed-based dFC analysis between the two groups.</p>
<p>To investigate the relationship between dALFF and dFC, a correlation analysis was carried out. This analysis aimed to explore how changes in spontaneous brain activity (as measured by dALFF) are related to alterations in functional interactions between regions (as measured by dFC). Understanding this relationship is crucial because it can provide insights into the underlying neural mechanisms and how different aspects of brain function are interconnected.</p>
<p>Additionally, the relationship between these neural alterations and the duration of <italic>Pingju</italic> training was explored through a correlation analysis to assess the association between the training duration and the observed changes in dALFF and dFC in the identified brain regions. This analysis aimed to determine whether longer training periods were linked to more significant neural changes. The significance level for these analyses was adjusted using the Bonferroni correction, with a threshold set at <italic>p</italic> &#x003C; 0.05.</p>
</sec>
<sec id="S2.SS7">
<title>Validation analysis</title>
<p>To ensure the robustness of our findings, we conducted a validation analysis using varying parameters for window length and step size to assess the consistency of our primary results. Specifically, we analyzed the data using four combinations: (1) a window length of 30 TRs with a step size of 1 TR, (2) a window length of 70 TRs with a step size of 1 TR, (3) a window length of 50 TRs with a step size of 5 TRs, and (4) a window length of 50 TRs with a step size of 10 TRs. We extracted the mean dALFF and dFC values from the significant ROIs identified in the primary results using these alternative parameters. Subsequently, a general linear model was applied to compare the dALFF and dFC values between groups, with gender and age included as covariates. Additionally, partial correlation analyses were performed to investigate the relationships both within dALFF and dFC and between these neural changes and the duration of training, controlling for age and sex.</p>
</sec>
</sec>
<sec id="S3" sec-type="results">
<title>Results</title>
<sec id="S3.SS1">
<title>Demographics of the participants</title>
<p>One <italic>Pingju</italic> actor was excluded from the study due to excessive head motion (mean FD &#x003E; 0.3 mm). The final sample consisted of 19 <italic>Pingju</italic> actors and 20 untrained subjects. Gender difference was assessed using a chi-square test, while age difference was evaluated with a two-sample <italic>t</italic>-test. The results showed that the two groups were well-matched in terms of gender (9 males in the <italic>Pingju</italic> actor group, 10 males in the untrained group, <italic>p</italic> = 0.98), handedness (all participants were right-handed), and age (30.47 &#x00B1; 2.29 years for the <italic>Pingju</italic> actors, 29.10 &#x00B1; 4.82 years for the untrained subjects, <italic>p</italic> = 0.27). Additionally, the <italic>Pingju</italic> actors had an average of 23.53 &#x00B1; 1.43 years of training. The detailed demographic data are presented in <xref ref-type="table" rid="T1">Table 1</xref>.</p>
<table-wrap position="float" id="T1">
<label>TABLE 1</label>
<caption><p>Demographic information of professional <italic>Pingju</italic> actors and untrained subjects.</p></caption>
<table cellspacing="5" cellpadding="5" frame="box" rules="all">
<thead>
<tr>
<td valign="top" align="left" style="color:#ffffff;background-color: #7f8080;">Variables</td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;">Actors</td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;">Untrained subjects</td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"><italic>p</italic>-value</td>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Gender (male/female)</td>
<td valign="top" align="center">9/10</td>
<td valign="top" align="center">10/10</td>
<td valign="top" align="center">0.98</td>
</tr>
<tr>
<td valign="top" align="left">Age (years)</td>
<td valign="top" align="center">30.47 &#x00B1; 2.29</td>
<td valign="top" align="center">29.10 &#x00B1; 4.82</td>
<td valign="top" align="center">0.27</td>
</tr>
<tr>
<td valign="top" align="left">Handedness (right/left)</td>
<td valign="top" align="center">20/0</td>
<td valign="top" align="center">20/0</td>
<td valign="top" align="center">&#x2013;</td>
</tr>
<tr>
<td valign="top" align="left">Years of training</td>
<td valign="top" align="center">23.53 &#x00B1; 1.43</td>
<td valign="top" align="center">&#x2013;</td>
<td valign="top" align="center">&#x2013;</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn><p>The <italic>p</italic> values were obtained by a two-sample <italic>t</italic>-test for age, and a chi-square test for gender. SD, standard deviation.</p></fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="S3.SS2">
<title>Differences in dALFF and dFC</title>
<p>The dALFF analysis revealed significant differences in spontaneous local brain activity between the two groups, with professional <italic>Pingju</italic> actors showing significantly lower dALFF in the right lingual gyrus compared to untrained individuals (<italic>p</italic> &#x003C; 0.05, cluster-level FWE correction; <xref ref-type="fig" rid="F1">Figure 1</xref> and <xref ref-type="table" rid="T2">Table 2</xref>). For the subsequent dFC analysis, the right lingual gyrus was used as the seed region. The results indicated that professional <italic>Pingju</italic> actors showed increased dFC between the right lingual gyrus and several regions, including the bilateral cerebellum, and bilateral midbrain/red nucleus/thalamus, compared to untrained individuals (<italic>p</italic> &#x003C; 0.05, cluster-level FWE correction; <xref ref-type="fig" rid="F2">Figure 2</xref> and <xref ref-type="table" rid="T2">Table 2</xref>).</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption><p>Dynamic ALFF changes in professional <italic>Pingju</italic> actors. <bold>(A)</bold> Brain regions showing significantly lower dALFF in professional <italic>Pingju</italic> actors compared to untrained subjects (cluster-level FWE <italic>p</italic> &#x003C; 0.05), particularly in the right lingual gyrus (highlighted in orange-yellow). <bold>(B)</bold> Violin plots comparing the mean dALFF values in the right lingual gyrus between professional <italic>Pingju</italic> actors and untrained subject. Professional <italic>Pingju</italic> actors show significantly lower dALFF than untrained subjects (<italic>p</italic> &#x003C; 0.001). Horizontal lines indicate median and quartiles. Abbreviations: dALFF, dynamic amplitude of low-frequency fluctuation; L, left; R, right.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fnins-18-1477181-g001.tif"/>
</fig>
<table-wrap position="float" id="T2">
<label>TABLE 2</label>
<caption><p>The brain regions with significant dALFF and dFC difference in professional <italic>Pingju</italic> actors.</p></caption>
<table cellspacing="5" cellpadding="5" frame="box" rules="all">
<thead>
<tr>
<td valign="top" align="left" style="color:#ffffff;background-color: #7f8080;">Brain regions</td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;">Cluster Size<break/> (Voxels)</td>
<td valign="top" align="center" colspan="3" style="color:#ffffff;background-color: #7f8080;">Peak MNI coordinate</td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;">Peak <italic>T</italic></td>
</tr>
<tr>
<td valign="top" align="left" style="color:#ffffff;background-color: #7f8080;"></td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"></td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"><italic>x</italic></td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"><italic>y</italic></td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"><italic>z</italic></td>
<td valign="top" align="center" style="color:#ffffff;background-color: #7f8080;"></td>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left" style="background-color: #dcdcdc;" colspan="6"><bold><italic>dALFF alteration</italic></bold></td>
</tr>
<tr>
<td valign="top" align="left">Right lingual gyrus</td>
<td valign="top" align="center">347</td>
<td valign="top" align="center">21</td>
<td valign="top" align="center">&#x2212;48</td>
<td valign="top" align="center">&#x2212;18</td>
<td valign="top" align="center">4.39</td>
</tr>
<tr>
<td valign="top" align="left" style="background-color: #dcdcdc;" colspan="6"><bold><italic>dFC alteration</italic></bold></td>
</tr>
<tr>
<td valign="top" align="left">Bilateral cerebellum</td>
<td valign="top" align="center">96</td>
<td valign="top" align="center">3</td>
<td valign="top" align="center">&#x2212;72</td>
<td valign="top" align="center">&#x2212;27</td>
<td valign="top" align="center">3.98</td>
</tr>
<tr>
<td valign="top" align="left">Bilateral midbrain/<break/> red nucleus/thalamus</td>
<td valign="top" align="center">146</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x2212;15</td>
<td valign="top" align="center">&#x2212;6</td>
<td valign="top" align="center">4.97</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn><p>dALFF, the dynamic amplitude of low-frequency fluctuation; dFC, the dynamic functional connectivity; MNI, Montreal Neurological Institute; <italic>T</italic>, <italic>t</italic>-statistic.</p></fn>
</table-wrap-foot>
</table-wrap>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption><p>Dynamic FC changes in professional <italic>Pingju</italic> actors using the right lingual gyrus as the seed region. <bold>(A)</bold> Brain regions showing significantly increased dFC in professional <italic>Pingju</italic> actors compared to untrained subjects (cluster-level FWE <italic>p</italic> &#x003C; 0.05). The regions include the bilateral cerebellum and bilateral midbrain/red nucleus/thalamus (highlighted in orange-yellow). <bold>(B)</bold> Violin plots comparing the mean dFC values between the right lingual gyrus and the bilateral cerebellum (left graph), and between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus (right graph). Professional <italic>Pingju</italic> actors show significantly higher dFC in both comparisons (<italic>p</italic> &#x003C; 0.001). Horizontal lines indicate median and quartiles. Abbreviations: dFC, the dynamic functional connectivity; L, left; R, right.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fnins-18-1477181-g002.tif"/>
</fig>
</sec>
<sec id="S3.SS3">
<title>Correlation analysis</title>
<p>The correlation analysis examining the relationship between dALFF and dFC revealed significant findings. As shown in <xref ref-type="fig" rid="F3">Figure 3</xref>, the dALFF of the right lingual gyrus was negatively correlated with the dFC between the right lingual gyrus and the cerebellum (<italic>r</italic> = &#x2212;0.52, <italic>p</italic> &#x003C; 0.001); similarly, it was also negatively correlated with the dFC between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus (<italic>r</italic> = &#x2212;0.43, <italic>p</italic> = 0.007).</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption><p>The relationship between dALFF and dFC in the right lingual gyrus. <bold>(A)</bold> Scatter plot showing the negative correlation between dALFF in the right lingual gyrus and dFC between the right lingual gyrus and the bilateral cerebellum (<italic>r</italic> = &#x2013;0.52, <italic>p</italic> &#x003C; 0.001). <bold>(B)</bold> Scatter plot showing the negative correlation between dALFF in the right lingual gyrus and dFC between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus (<italic>r</italic> = &#x2013;0.43, <italic>p</italic> = 0.007). Abbreviations: dALFF, the dynamic amplitude of low-frequency fluctuation; dFC, the dynamic functional connectivity.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fnins-18-1477181-g003.tif"/>
</fig>
<p>Further analysis explored the relationships between these neural alterations and the duration of <italic>Pingju</italic> training. After applying the Bonferroni correction (<italic>p</italic> &#x003C; 0.05/3 = 0.0167), a significant positive correlation was found between the dFC between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus and the duration of training (<italic>r</italic> = 0.64, <italic>p</italic> = 0.003; <xref ref-type="fig" rid="F4">Figure 4</xref>). However, the dALFF of the right lingual gyrus and the dFC between the right lingual gyrus and bilateral cerebellum were not significantly correlated with the duration of training.</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption><p>Correlation between training duration and dFC between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus. Scatter plot showing the positive correlation between the duration of <italic>Pingju</italic> training and dFC between the right lingual gyrus and the bilateral midbrain/red nucleus/thalamus (<italic>r</italic> = 0.64, <italic>p</italic> = 0.003). dFC, the dynamic functional connectivity.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fnins-18-1477181-g004.tif"/>
</fig>
</sec>
<sec id="S3.SS4">
<title>Validation analysis</title>
<p>Our validation analysis demonstrated the consistency of our primary findings across a range of analytical conditions. When we examined dALFF and dFC values in significant ROIs, the results were robust across all testing configurations: (1) a window length of 30 TRs with a step size of 1 TR, (2) a window length of 70 TRs with a step size of 1 TR, (3) a window length of 50 TRs with a step size of 5 TRs, and (4) a window length of 50 TRs with a step size of 10 TRs. These varied settings confirmed that the significant differences initially observed in the standard setting of 50 TRs and 1 TR step size persisted. Additionally, the partial correlation analyses reinforced the relationships within dALFF and dFC and between these neural measures and the duration of <italic>Pingju</italic> training, remaining stable regardless of the analytical parameters used. For detailed results, please see <xref ref-type="supplementary-material" rid="DS1">Supplementary Figures 1</xref>&#x2013;<xref ref-type="supplementary-material" rid="DS1">4</xref>.</p>
</sec>
</sec>
<sec id="S4" sec-type="discussion">
<title>Discussion</title>
<p>To the best of our knowledge, this study represents the first attempt to investigate the effects of long-term <italic>Pingju</italic> training on dynamic brain activity and connectivity. We found that professional <italic>Pingju</italic> actors exhibited significantly lower dALFF in the right lingual gyrus compared to untrained individuals. Additionally, the actors showed increased dFC between the right lingual gyrus and both the bilateral cerebellum and the bilateral midbrain/red nucleus/thalamus. These findings suggest that extensive training in <italic>Pingju</italic> may induce neuroplastic changes by altering local brain activity and the FC between brain regions, highlighting the importance of understanding how specialized training in the arts can influence brain function.</p>
<p>Our study identified a significant decrease in the dALFF in the right lingual gyrus among professional <italic>Pingju</italic> actors compared with untrained individuals. The right lingual gyrus is predominantly associated with visual processing (<xref ref-type="bibr" rid="B5">Bogousslavsky et al., 1987</xref>; <xref ref-type="bibr" rid="B39">Palejwala et al., 2021</xref>; <xref ref-type="bibr" rid="B7">Chai et al., 2024</xref>), including the interpretation of complex visual stimuli such as shapes and spatial orientation (<xref ref-type="bibr" rid="B19">Hill et al., 2019</xref>; <xref ref-type="bibr" rid="B3">Baltaretu et al., 2021</xref>). In <italic>Pingju</italic>, performers must develop exceptional visual-motor coordination to execute precise movements and interpret visual cues on stage. This extensive training beginning at a young age may result in the enhanced specialization and efficiency of the neural circuits within the lingual gyrus. The observed decrease in dALFF may reflect a more streamlined neural processing mechanism in this region, as the brain adapts to the repetitive and intensive visual tasks required by <italic>Pingju</italic>. This reduction indicates that the actors&#x2019; brains have become more efficient at handling visual information, reducing the need for heightened spontaneous neural activity. Similar patterns of neural adaptation have been reported in studies on neuroplasticity related to musical training, where reduced mean diffusivity in the lingual gyrus was observed (<xref ref-type="bibr" rid="B38">Olszewska et al., 2021</xref>). The decrease in dALFF within the right lingual gyrus observed in our current study may, therefore, suggest a similar neuroplastic adaptation, optimizing the brain&#x2019;s response to the specific demands of <italic>Pingju</italic> performance, such as visual-motor integration and spatial awareness.</p>
<p>The results of the dFC analysis revealed that professional <italic>Pingju</italic> actors exhibited increased dFC between the right lingual gyrus and bilateral cerebellum, as well as the bilateral midbrain/red nucleus/thalamus. This enhanced connectivity illustrates the intricate relationship between motor coordination, sensory processing, and cognitive functions (<xref ref-type="bibr" rid="B18">Higashionna et al., 2017</xref>; <xref ref-type="bibr" rid="B40">Pastor-Cerezuela et al., 2020</xref>), which is crucial for the highly specialized and demanding performance in <italic>Pingju</italic>. The cerebellum plays a pivotal role in motor control, balance, and coordination (<xref ref-type="bibr" rid="B31">Manto et al., 2012</xref>; <xref ref-type="bibr" rid="B44">Rudolph et al., 2023</xref>). Increased connectivity between the right lingual gyrus and the bilateral cerebellum suggests that <italic>Pingju</italic> training strengthens the integration of visual and motor functions. This enhancement likely supports the precise and fluid movements required on stage, allowing performers to execute complex sequences with greater accuracy and timing. Similar neuroplastic adaptations have been observed in other artistic professions, such as musicians and dancers, where extensive training also leads to enhanced FC between motor, sensory, and cognitive brain regions (<xref ref-type="bibr" rid="B45">Schlaug, 2001</xref>; <xref ref-type="bibr" rid="B12">Eierud et al., 2023</xref>). Additionally, the midbrain, red nucleus, and thalamus are involved in sensorimotor integration, motor coordination, and alertness (<xref ref-type="bibr" rid="B55">Yanaka et al., 2010</xref>; <xref ref-type="bibr" rid="B4">Basile et al., 2021</xref>; <xref ref-type="bibr" rid="B49">Wolff et al., 2021</xref>; <xref ref-type="bibr" rid="B32">Mao et al., 2022</xref>). The increased dFC between the right lingual gyrus and these regions indicates a more robust communication network supporting the sensory-motor demands of <italic>Pingju</italic>. The red nucleus, as part of the extrapyramidal system, is crucial for fine motor control (<xref ref-type="bibr" rid="B36">Ogut et al., 2023</xref>), and its enhanced connectivity with the right lingual gyrus may facilitate the intricate hand and body movements characteristic of <italic>Pingju</italic> performance. This finding aligns with research on ballet dancers, where enhanced connectivity in the extrapyramidal system has been linked to the refined motor skills required for complex dance routines (<xref ref-type="bibr" rid="B6">Burzynska et al., 2017</xref>). Overall, these results demonstrate how <italic>Pingju</italic> training enhances FC in brain regions involved in motor coordination and sensory integration, reflecting specialized neuroplastic adaptations that support the precise movements and complex performances required in this art form.</p>
<p>The correlation analysis revealed significant neural adaptations associated with long-term <italic>Pingju</italic> training. Specifically, the dALFF of the right lingual gyrus was negatively correlated with the dFC between the right lingual gyrus and both the cerebellum and the midbrain/red nucleus/thalamus. These findings suggest that lower spontaneous activity in the right lingual gyrus is linked to higher FC with these regions, indicating more efficient neural processing for the complex visual and motor tasks required in <italic>Pingju</italic>. Additionally, the duration of training positively correlates with the dFC between the right lingual gyrus and the midbrain/red nucleus/thalamus, highlighting enhanced connectivity with extended practice. These findings emphasize the adaptive changes in brain function due to specialized, long-term artistic training, enhancing neural efficiency and integration in regions critical for <italic>Pingju</italic> performance.</p>
<p>Several limitations of this study must be acknowledged. First, the sample size was relatively small, with only 20 professional <italic>Pingju</italic> actors and 20 untrained individuals. This limited sample size may affect the generalizability of the results to a broader population of opera performers or other individuals engaged in similar artistic training. Second, the cross-sectional design restricts our ability to draw causal conclusions about the effects of long-term <italic>Pingju</italic> training on brain function. Longitudinal studies are needed to better understand the causal relationships and the progression of neuroplastic changes over time. Finally, we did not collect data on several potential confounding factors, such as lifestyle differences, stress levels, or other forms of physical or cognitive training. These factors could independently influence brain activity and connectivity, potentially contributing to the observed differences in dALFF and dFC between <italic>Pingju</italic> actors and untrained subjects.</p>
</sec>
<sec id="S5" sec-type="conclusion">
<title>Conclusion</title>
<p>In conclusion, our study demonstrated that long-term <italic>Pingju</italic> training induces significant neuroplastic changes, as evidenced by altered dALFF and dFC in the brain. Specifically, professional <italic>Pingju</italic> actors exhibited reduced dALFF in the right lingual gyrus, coupled with increased dFC between the right lingual gyrus and both the bilateral cerebellum and the bilateral midbrain/red nucleus/thalamus. These findings contribute to our understanding of how specialized, long-term artistic training can reshape brain function, reinforcing the potential of cultural practices like <italic>Pingju</italic> to modulate neural architecture. Future research should explore the cognitive and emotional implications of these neural changes, elucidating the impact of professional artistic training on brain health and cognitive functions.</p>
</sec>
</body>
<back>
<sec id="S6" sec-type="data-availability">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="supplementary-material" rid="DS1">Supplementary material</xref>, further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec id="S7" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>The studies involving humans were approved by the Ethics Committee of Tianjin Medical University General Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.</p>
</sec>
<sec id="S8" sec-type="author-contributions">
<title>Author contributions</title>
<p>FZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing &#x2013; original draft. LS: Conceptualization, Formal analysis, Investigation, Validation, Writing &#x2013; review and editing. YC: Investigation, Writing &#x2013; review and editing, Data curation, Software, Visualization. SW: Investigation, Writing &#x2013; review and editing, Methodology. XW: Investigation, Conceptualization, Supervision, Writing &#x2013; original draft. YZ: Investigation, Validation, Visualization, Writing &#x2013; review and editing. JX: Investigation, Visualization, Writing &#x2013; review and editing. ZZ: Visualization, Supervision, Writing &#x2013; original draft. ML: Methodology, Project administration, Resources, Writing &#x2013; review and editing. WC: Writing &#x2013; review and editing, Investigation, Supervision. QA: Writing &#x2013; review and editing, Conceptualization, Formal analysis. DZ: Supervision, Visualization, Writing &#x2013; original draft. FL: Supervision, Funding acquisition, Resources, Writing &#x2013; review and editing. CW: Conceptualization, Formal analysis, Writing &#x2013; review and editing. FL: Funding acquisition, Resources, Supervision, Writing &#x2013; review and editing.</p>
</sec>
<sec id="S9" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Special Research Fund for Central Universities, Peking Union Medical College (3332023098), the Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-001A), the Natural Science Foundation of Tianjin (22JCQNJC01450), and the TianHe Qingsuo Open Research Fund of TSYS in 2022 and NSCCTJ (P-THQS-22-PY-No.0004).</p>
</sec>
<sec id="S10" sec-type="COI-statement">
<title>Conflict of interest</title>
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