Journal of Neuroscience Methods最新文献

{"title":"Immuno-laser capture microdissection of perfusion-fixed mouse brain tissue coupled to RNA-seq.","authors":"Shujun Ge, Wenqi Gan, Keaton Karlinsey, Beiyan Zhou, Joel S Pachter","doi":"10.1016/j.jneumeth.2025.110548","DOIUrl":"10.1016/j.jneumeth.2025.110548","url":null,"abstract":"<p><strong>Background: </strong>Laser capture microdissection (LCM), when combined with immunostaining (immuno-LCM) and coupled to RNA profiling, provides a means for in situ transcriptomic interrogation of complex tissues. However, preserving RNA integrity through the multiple harsh steps of immuno-LCM has proved challenging, greatly limiting the potential for high-resolution spatial analysis of global gene expression.</p><p><strong>New method: </strong>Here, we describe a protocol whereby perfusion fixation and subsequent ex vivo post-fixation of mouse brain with paraformaldehyde, followed by protease digestion of immuno-LCM-acquired material from brain sections, allows for isolation of RNA of relatively high integrity that is amenable to RNA-seq with minimal technical variability.</p><p><strong>Results: </strong>The individual steps of the fixation/immuno-LCM protocol were evaluated for their respective effects on RNA integrity and found not to produce significant compromise, as judged by RIN values determined using a Bioanalyzer or Tape Station. Utilizing the fixation/immuno-LCM protocol to assess gene expression from targeted brain microvascular tissue further showed high reproducibility in both qRT-PCR and RNA-seq analysis, as determined by interpolation and Pearson Correlation, respectively, with the latter detecting ∼ 22,000 genes, including those in the established blood-brain barrier transcriptome.</p><p><strong>Comparison with existing methods: </strong>To date, there has been no detailed analysis of how fixation combined with immuno-LCM impacts RNA integrity and transcriptomic analysis.</p><p><strong>Conclusions: </strong>The rigorous analyses performed demonstrate that paraformaldehyde fixation - which covalently cross-links RNA and protein - can be reversed without significant damage to RNA integrity, and can be combined with immuno-LCM to enable high-resolution spatial analysis of global gene expression.</p>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":" ","pages":"110548"},"PeriodicalIF":2.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12377190/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144804295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel device for studying temperature and touch interactions.","authors":"Juan Carlos Ramirez, Jose Vergara, Jing Lin, Jian Chen, Jeffrey M Yau","doi":"10.1016/j.jneumeth.2025.110547","DOIUrl":"10.1016/j.jneumeth.2025.110547","url":null,"abstract":"<p><strong>Background: </strong>Existing methods to study the effects of skin temperature on mechanical touch perception range from large hot plates, water baths, or bulky, water-controlled thermal contactors which have limited range and resolution. The limited capabilities of these methods prevent the study of thermo-tactile interactions at the finger level in a flexible and precisely controlled manner.</p><p><strong>New method: </strong>Here, we combine small Proportional-Integral-Derivative (PID)-controlled Peltier elements with a calibrated shaker motor for a novel thermo-tactile stimulus delivery system capable of precisely controlling temperature and vibrotactile stimulation to the fingertip. This novel system enables parallel control of mechanical stimulation and thermal stimulation at congruent skin sites of the fingertip. Alternative thermoelectric elements and mechanical actuators could be used in our systems modular configuration.</p><p><strong>Results: </strong>Our thermo-tactile delivery system can simultaneously deliver precise and stable vibrotactile and thermal stimuli over 30-250 Hz and 20-40°C, respectively, at the fingertip. We validated our system in psychophysical tests and reproduced the established finding that vibration detection thresholds vary according to temperature.</p><p><strong>Comparison with existing method(s): </strong>Unlike our system, existing methods to study thermo-tactile interactions are restricted to testing skin regions larger than the fingertip or they use tactile probes on the fingertips that are not thermally controlled.</p><p><strong>Conclusions: </strong>Our system represents a novel strategy for combining thermoelectric modules with mechanical actuation to study thermo-tactile interactions at mechanoreceptor-rich fingertips.</p>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":" ","pages":"110547"},"PeriodicalIF":2.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144775588","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation of structure tensor analysis for orientation estimation in brain tissue microscopy.","authors":"Bryson Gray, Andrew W Smith, Allan MacKenzie-Graham, David W Shattuck, Daniel J Tward","doi":"10.1016/j.jneumeth.2025.110539","DOIUrl":"10.1016/j.jneumeth.2025.110539","url":null,"abstract":"<p><strong>Background: </strong>Accurate localization of white matter pathways using diffusion MRI is critical to investigating brain connectivity, but the accuracy of current methods is not thoroughly understood. A fruitful approach to validating accuracy is to consider microscopy data that have been co-registered with MRI of post mortem samples. In this setting, structure tensor analysis is a standard approach to computing local orientations. However, structure tensor analysis itself has not been well-validated and is subject to uncertainty in its angular resolution, and selectivity to specific spatial scales.</p><p><strong>New method: </strong>Here, we conducted a simulation study to investigate the accuracy of using structure tensors to estimate the orientations of fibers arranged in configurations with and without crossings.</p><p><strong>Results: </strong>We examined a range of simulated conditions, with a focus on investigating the method's behavior on images with anisotropic resolution, which is particularly common in microscopy data acquisition. We also analyzed 2D and 3D optical microscopy data.</p><p><strong>Comparison with existing methods: </strong>Our results show that parameter choice in structure tensor analysis has relatively little effect on accuracy for estimating single orientations, although accuracy decreases with increasing resolution anisotropy. On the other hand, when estimating the orientations of crossing fibers, the choice of parameters becomes critical, and poor choices result in orientation estimates that are essentially random.</p><p><strong>Conclusions: </strong>This work provides a set of recommendations for researchers seeking to apply structure tensor analysis effectively in the study of axonal orientations in brain imaging data and quantifies the method's limitations, particularly in the case of anisotropic data.</p>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":" ","pages":"110539"},"PeriodicalIF":2.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144753616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparing Human Annotation and Machine Learning Models for Optimizing Zebrafish Behavioral Classification in Seizure Analysis.","authors":"Barbara D Fontana, Camilla W Pretzel, Mariana L Müller, Kimberly Fontoura, Khadija A Mohammed, Eduarda T Saccol, Falco L Gonçalves, Angela E Uchoa, Carolina C Jardim, Isabella P Silva, Rossano M Silva, Hevelyn S Moraes, Cássio M Resmim, Julia Canzian, Denis B Rosemberg","doi":"10.1016/j.jneumeth.2025.110603","DOIUrl":"https://doi.org/10.1016/j.jneumeth.2025.110603","url":null,"abstract":"<p><strong>Background: </strong>Accurate and scalable behavioral annotation remains a challenge in behavioral neuroscience. Manual scoring is time-consuming, variable across annotators, and may overlook transient behaviors critical for phenotyping. By learning from annotated datasets, supervised machine learning (ML) enables automated classification of behavior with high consistency and reduced bias.</p><p><strong>New method: </strong>We benchmarked five supervised ML algorithms, Random Forest, XGBoost, Support Vector Machine, k-Nearest Neighbors, and Multilayer Perceptron (MLP), and compared data against expert human annotations of seizure-like behaviors in adult zebrafish. Twelve trained raters annotated over 43,000 video frames, enabling direct comparison of model performance with human annotation. After frame-level analysis, we also applied behavior-informed filters and then evaluated block-level temporal aggregation.</p><p><strong>Results: </strong>Annotation variability was driven by behavioral complexity, with ambiguous behaviors showing the lowest agreement. Random Forest, XGBoost, and MLP achieved the highest accuracy and post-processing based on posture and velocity improved classification by filtering false positives. Block-level aggregation enhanced accuracy through temporal smoothing but masked short-lived behaviors critical for detecting subtle phenotypes.</p><p><strong>Comparison with existing methods: </strong>Most zebrafish seizure studies rely on manual scoring or single-model ML applications. Direct comparisons between multiple ML algorithms and human annotations are rare. Our study uniquely integrates large-scale manual scoring with model benchmarking and temporal resolution strategies, offering insight into reproducibility and scalability in behavioral phenotyping.</p><p><strong>Conclusions: </strong>This study advances automated behavioral analysis in zebrafish by demonstrating the strengths and limitations of machine learning compared to human annotation, and emphasizes how choices in temporal resolution and behavioral classification influence reproducibility and interpretability.</p>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":" ","pages":"110603"},"PeriodicalIF":2.3,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of tissue storage time on immunodetection of c-Fos and GAD67 in the rat brain","authors":"Stoyan Dimitrov ,&nbsp;Xia Shan ,&nbsp;Jan Born ,&nbsp;Marion Inostroza","doi":"10.1016/j.jneumeth.2025.110602","DOIUrl":"10.1016/j.jneumeth.2025.110602","url":null,"abstract":"<div><h3>Background</h3><div>Activity-dependent markers such as c-Fos, a rapid indicator of neuronal activation, and GAD67, an enzyme essential for GABA synthesis in inhibitory neurons, are extensively employed to elucidate neural circuit dynamics. Given that many studies span extended periods with multiple experimental groups, it is crucial to ensure long-term storage of non-frozen brain tissue does not compromise immunodetection.</div></div><div><h3>New method</h3><div>Here, we evaluated the impact of storage duration on the immunodetection of c-Fos and GAD67 in rat brains. Intact brains, fixed in paraformaldehyde, were stored at 4 °C in phosphate-buffered saline with sodium azide to prevent bacterial growth. Brains were assessed at two storage durations - short (1.5 months) and prolonged (10 months). Brain sections were immunostained for c-Fos and GAD67 and imaged by confocal microscopy.</div></div><div><h3>Results</h3><div>We observed robust c-Fos immunoreactivity across multiple regions of the medial prefrontal cortex, hippocampus, and neocortex, with no significant differences attributable to storage duration. Additionally, quantifications of GAD67-positive cells and cells co-labeled for c-Fos/ GAD67 confirmed that immunodetection of inhibitory neurons remains intact when whole brains are stored for up to 10 months. In contrast, prolonged storage of brain slices strongly reduced c-Fos, but increased GAD67 staining.</div></div><div><h3>Comparison with existing method(s)</h3><div>The stability of c-Fos and GAD67 in tissue stored long-term at 4 °C remains untested.</div></div><div><h3>Conclusions</h3><div>These findings underscore that whereas intact brains can be safely stored for prolonged periods at 4°C without compromising antigenicity, brain slices are highly susceptible to storage-induced deterioration - insights important for planning and interpreting immunohistochemical studies in neuroscience.</div></div>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":"425 ","pages":"Article 110602"},"PeriodicalIF":2.3,"publicationDate":"2025-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145344947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an in vitro fibrotic scar model of spinal cord injury using macromolecular crowding","authors":"Sorour Nemati ,&nbsp;Alanna Stanley ,&nbsp;Michelle Kilcoyne ,&nbsp;Dimitrios Zeugolis ,&nbsp;Siobhan S. McMahon","doi":"10.1016/j.jneumeth.2025.110601","DOIUrl":"10.1016/j.jneumeth.2025.110601","url":null,"abstract":"<div><h3>Background</h3><div>Spinal cord injury (SCI) results in a cascade of cellular and molecular events that lead to permanent tissue damage and functional impairment. A key consequence of this injury is the formation of both glial and fibrotic scars, which pose significant barriers to regeneration. The fibrotic scar that forms following SCI remains a significant therapeutic challenge. One major obstacle in developing anti-fibrotic compounds is the absence of a comprehensive <em>in vitro</em> screening system.</div></div><div><h3>New method</h3><div>In this study, we employed a macromolecular crowding (MMC) technique to accelerate ECM deposition. Leptomeningeal (LPG) cells were cultured in media supplemented with the MMC Ficoll (FC). To mimic the injury environment <em>in vivo</em>, the cells were exposed to either physical or chemical injury.</div></div><div><h3>Results</h3><div>The growth and metabolic activity of the LPG cells remained unchanged under these different injuries and treatments. Groups supplemented with the MMC FC exhibited higher deposition of ECM proteins involved in fibrotic scar formation, including fibronectin, collagen IV, collagen I, and laminin, compared to those without FC.</div></div><div><h3>Comparison with existing methods</h3><div>A key limitation of conventional cell culture in aqueous media is its clear difference from the naturally ‘crowded’ tissue environment, resulting in a slow rate of ECM protein deposition. Using the MMC approach, we successfully accelerated ECM protein deposition within an <em>in vitro</em> model of the fibrotic scar.</div></div><div><h3>Conclusions</h3><div>Supplementing LPG culture media with MMCs can effectively mimic the fibrotic scar environment, providing a valuable refinement in developing SCI <em>in vitro</em> models for drug screening and therapeutic applications.</div></div>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":"424 ","pages":"Article 110601"},"PeriodicalIF":2.3,"publicationDate":"2025-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145292470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Methodological determinants of signal quality in electrobulbogram recordings","authors":"Frans Nordén ,&nbsp;Irene Zanettin ,&nbsp;Tora Olsson ,&nbsp;Artin Arshamian ,&nbsp;Mikael Lundqvist ,&nbsp;Fahimeh Darki ,&nbsp;Johan N. Lundström","doi":"10.1016/j.jneumeth.2025.110598","DOIUrl":"10.1016/j.jneumeth.2025.110598","url":null,"abstract":"<div><div>The electrobulbogram (EBG) is a new, non-invasive method for measuring the functional activity of the human olfactory bulb (OB). To date, the EBG has been used to assess how the OB process odor identity, valence, intensity, and it has shown promise as an early biomarker for Parkinson’s disease. However, current implementation of the EBG method depends on several methodological components, including subject specific co-registration of electrode positions through neuronavigation and EEG source reconstruction, which may limit accessibility for many research groups. In this study, we test the quality and reliability of the OB signal under different configurations to potentially remedy this. Specifically, we compare six EBG setups that vary in the use of subject-specific T1 scans versus a template head model, co-registered versus template electrode positions, and individualized versus template-based OB location. Our results indicate that strongest EBG signals are obtained when using subject-specific T1 scans in combination with co-registered electrode positions. However, we obtained significant EBG activity even when using a fully template-based configuration. Our anatomical analysis of OB location of 941 individuals reveals that in 86 % of cases, the OB is centered within the spatial resolution bounds of the EEG source dipole, supporting the feasibility of detecting olfactory bulb signals without precise individual anatomical mapping using template coordinates. These findings suggest that while subject-specific configurations enhance signal quality, the EBG method remains robust enough to yield meaningful results even with less complex setups. This enables a broader adoption of the EBG method in both clinical and research settings.</div></div>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":"424 ","pages":"Article 110598"},"PeriodicalIF":2.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145267863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sensitivity analysis of the balloon model parameters in functional near-infrared spectroscopy simulation","authors":"Murad Althobaiti","doi":"10.1016/j.jneumeth.2025.110599","DOIUrl":"10.1016/j.jneumeth.2025.110599","url":null,"abstract":"<div><h3>Background</h3><div>Accurate modeling of the hemodynamic response is critical for fNIRS data interpretation. While the Balloon model is a cornerstone for this, the quantitative impact of its key parameters on the fNIRS signal, particularly in the presence of realistic artifacts, remains under-characterized.</div></div><div><h3>New method</h3><div>We developed an end-to-end fNIRS simulation pipeline. It incorporates a neural activity model, the Balloon model for hemodynamics, convolution for signal generation, and realistic motion, cardiac, and respiratory artifacts. We performed a sensitivity analysis by systematically varying Grubb's exponent (α) and transit time (τ).</div></div><div><h3>Results</h3><div>Both α and τ significantly influence the simulated fNIRS response. α shows a non-linear relationship with peak amplitude, while τ has a more linear effect on signal timing. Regression models quantifying these effects demonstrated a strong statistical fit (p &lt; 0.05, R² &gt; 0.9 for α).</div></div><div><h3>Comparison with existing methods</h3><div>Unlike prior fMRI-focused studies, this is the first quantitative sensitivity analysis specifically for fNIRS signals that incorporates a realistic noise model. Our framework characterizes the forward model's behavior, providing parameter-specific insights not previously available for fNIRS simulations.</div></div><div><h3>Conclusions</h3><div>The fNIRS hemodynamic response is highly sensitive to the Balloon model's α and τ parameters. These findings highlight the importance of accounting for physiological variability in fNIRS analysis and provide a robust framework for generating synthetic data to test signal processing algorithms.</div></div>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":"424 ","pages":"Article 110599"},"PeriodicalIF":2.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145267857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing attentiveness and cognitive engagement across tasks using video-based action understanding in non-human primates","authors":"Sin-Man Cheung ,&nbsp;Adam Neumann ,&nbsp;Thilo Womelsdorf","doi":"10.1016/j.jneumeth.2025.110597","DOIUrl":"10.1016/j.jneumeth.2025.110597","url":null,"abstract":"<div><h3>Background</h3><div>Distractibility and attentiveness are cognitive states that are expressed through observable behavior, but how behavioral features can be used to quantify these cognitive states has remained poorly understood. Video-based analysis promises to be a versatile tool to quantify the behavioral features that reflect subject-specific distractibility and attentiveness and are diagnostic of cognitive states.</div></div><div><h3>New method</h3><div>We describe an analysis pipeline that classifies cognitive states using a 2-camera set-up for video-based estimation of attentiveness and screen engagement in nonhuman primates performing cognitive tasks. The procedure reconstructs 3D poses from 2D labeled DeepLabCut videos, reconstructs the head/yaw orientation relative to a task screen, and arm/hand/wrist engagements with task objects, to segment behavior into an attentiveness and engagement score.</div></div><div><h3>Results</h3><div>Performance of different cognitive tasks was robustly classified from video within a few frames, reaching &gt; 90 % decoding accuracy with ≤ 3 min long time segments. The analysis procedure allows adjusting thresholds for segmenting subject-specific movements for a time-resolved scoring of attentiveness and screen engagement.</div></div><div><h3>Comparison with existing methods</h3><div>Current methods also extract poses and segment action units; however, they haven't been combined into a framework that enables subject-adjusted thresholding for specific task contexts. This integration is needed for inferring cognitive state variables and differentiating performance across various tasks.</div></div><div><h3>Conclusion</h3><div>The proposed method integrates video segmentation, scoring of attentiveness and screen engagement, and classification of task performance at high temporal resolution. This integrated framework provides a tool for assessing attention functions from video.</div></div>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":"424 ","pages":"Article 110597"},"PeriodicalIF":2.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145267859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3D-Printable Non-invasive Head Immobilization System for Non-Human Primates.","authors":"Elia Shahbazi, Drew Nguyen, Tyler Swedan, Timothy Ma, Rosa Lafer-Sousa, Alvin Dinh, Reza Azadi, Amy M Ryan, Arash Afraz","doi":"10.1016/j.jneumeth.2025.110593","DOIUrl":"https://doi.org/10.1016/j.jneumeth.2025.110593","url":null,"abstract":"<p><strong>Background: </strong>Efficient head fixation is critical in primate behavioral experiments for accurate eye tracking and neural studies. Existing methods risk infection and bone necrosis due to their invasive nature.</p><p><strong>New method: </strong>We propose a custom-designed, 3D-printed non-invasive head immobilization system (NHIS) for macaques based on precise CT/MRI scans and integrated with our specialized software, FLoRIN, and Blender 3D. This NHIS is tailored for straightforward manufacturing and personalization and accommodates the necessary experimental equipment.</p><p><strong>Results: </strong>This innovative NHIS ensures reliable head stabilization while significantly reducing invasive procedure risks. The design simplifies the setup process, curtails infection risk, mitigates bone damage, and increases comfort for subjects.</p><p><strong>Comparison with existing method(s): </strong>Our NHIS surpasses previous invasive and non-invasive methods by removing the need for surgical implantation, extensive training, and discomfort. It is designed for ease of adjustment and fitting without sedation, enhancing adaptability and animal welfare.</p><p><strong>Conclusions: </strong>The NHIS signifies a significant advancement in non-invasive primate head fixation, providing a customizable, less invasive, and more reliable solution for research. Its potential for widespread adoption in diverse research scenarios promises to improve the subject well-being and the quality of data collected.</p>","PeriodicalId":16415,"journal":{"name":"Journal of Neuroscience Methods","volume":" ","pages":"110593"},"PeriodicalIF":2.3,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145274760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}