{"title":"Single-cell endogenous protein labeling via CRISPR-Cas9-mediated genome editing in the mouse brain.","authors":"Motokazu Uchigashima, Takayasu Mikuni","doi":"10.1007/s12565-025-00866-x","DOIUrl":"https://doi.org/10.1007/s12565-025-00866-x","url":null,"abstract":"<p><p>High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625288","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}
Kazunori Toida, Keita Satoh, Haruyo Yamanishi, Yukari Minami-Ogawa, Simone N T Kurial, Emi Kiyokage
{"title":"Correlative light and electron microscopy analysis for neurobiological applications.","authors":"Kazunori Toida, Keita Satoh, Haruyo Yamanishi, Yukari Minami-Ogawa, Simone N T Kurial, Emi Kiyokage","doi":"10.1007/s12565-025-00868-9","DOIUrl":"https://doi.org/10.1007/s12565-025-00868-9","url":null,"abstract":"<p><p>In this article, we will introduce the significance of correlative light and electron microscopy analyses, an integrated morphological analysis method that directly combines laser and electron microscopes. This protocol comprehensively instructs the appropriate methods for the visualization of genetically labeled neurons in mice using digitalized electron microscopes. We wish to consider with the readers the importance and functional potential of electron microscopic analysis for medical and biological applications, especially for neurobiological applications.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144590290","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":"Acceleration of axonal regeneration by GABA/Gly excitation.","authors":"Chitoshi Takayama, Tsukasa Yafuso","doi":"10.1007/s12565-025-00869-8","DOIUrl":"https://doi.org/10.1007/s12565-025-00869-8","url":null,"abstract":"<p><p>In the mature central nervous system (CNS), γ-aminobutyric acid (GABA) and glycine (Gly) are predominant inhibitory neurotransmitters that negatively regulate neural activities. In contrast, GABA mediates membrane potential depolarization during development, and GABA/Gly become excitatory after nerve injury because of the high intracellular Cl<sup>-</sup> concentration induced by low expression of K<sup>+</sup>, Cl<sup>-</sup> cotransporter 2 (KCC2), which transports Cl<sup>-</sup> out of neurons. Many studies have reported that during CNS development, GABAergic excitatory action might play crucial roles in neurogenesis through Ca<sup>2+</sup> influx. Nevertheless, its involvement in neurogenesis has not been proven because the CNS can develop normally without GABAergic signals. Recently, two research groups demonstrated that low level of KCC2 (i.e., GABA/Gly excitation) after nerve injury is involved in axonal regeneration and in enhancement of functional recovery. In this manuscript, we review GABA/Gly excitation and introduce recent findings describing its involvement in axonal regeneration.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144590289","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}
Sven Hildebrand, Johannes Franz, Shubharthi Sengupta, Anna Schueth, Andreas Herrler, Alard Roebroeck
{"title":"ht-MASH: a high-throughput, cost-effective, and robust protocol for microscopic 3D imaging of human angio- and cytoarchitecture in large human brain samples.","authors":"Sven Hildebrand, Johannes Franz, Shubharthi Sengupta, Anna Schueth, Andreas Herrler, Alard Roebroeck","doi":"10.1007/s12565-025-00859-w","DOIUrl":"https://doi.org/10.1007/s12565-025-00859-w","url":null,"abstract":"<p><p>The performance of many optical tissue clearing protocols has considerably improved in the last few years, so that now even notoriously difficult specimen such as highly myelinated human brain tissue can be rendered highly transparent. However, optical tissue clearing is still routinely performed on relatively small samples, especially in the case of the human brain. Recent advances in histological tissue processing now allow scaling up the clearing process considerably towards much larger samples. Yet so far, these methods can have considerable drawbacks in their feasibility to be implemented routinely, especially in smaller laboratories. Here, we present an updated version of our MASH protocol, which allows optical tissue clearing of very large human brain tissue samples and labelling of angio- and cytoarchitecture therein. This pipeline is cost-efficient and easy to implement, so that even smaller labs can apply it at scale. At the same time, the use of rapid prototyping using 3D printing to create custom clearing equipment is versatile enough to be adjusted to other optical tissue clearing methods than the one used in this study (e.g., aqueous methods such as CUBIC or other solvent-based methods of the DISCO family), sample sizes or tissue types. Our pipeline has, therefore, the potential to advance optical tissue clearing and labelling of large human tissue samples towards a more robust and routine implementation in the blooming field of 3D histology.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144582846","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":"Tracing of specific neural pathways in the rat brain using lentiviral vectors for retrograde gene transfer.","authors":"Saaya Akama, Yoshio Iguchi, Shigeki Kato, Kazuto Kobayashi","doi":"10.1007/s12565-025-00864-z","DOIUrl":"https://doi.org/10.1007/s12565-025-00864-z","url":null,"abstract":"<p><p>Understanding the structural and the functional organization of neural circuits in the brain is a fundamental goal of neuroscience. Lentiviral vectors for retrograde gene transfer transduce neurons through the entry from synaptic terminals and enable tracing and manipulation of neuronal populations of interests based on the synaptic connections. The highly efficient and neuron-specific retrograde gene transfer (NeuRet) vectors are derived from the pseudotyping of human immunodeficiency virus type 1-based vectors with fusion envelope glycoproteins. Viral RNA genome is reverse-transcribed and synthesized DNA is integrated into the host genomes, allowing stable and long-term expression of transgenes. Dorsal striatum, the input layer of the basal ganglia, integrates signals from various brain regions, including cerebral cortex, thalamus, and ventral midbrain, and plays a critical role in motor control, learning, and decision-making. Consequently, understanding the input and the output connectivity of the dorsal striatum is fundamental to revealing how circuits within the basal ganglia contribute to physiological and behavioral functions, and its impairments are related to neurological disorders. This paper outlines the procedures for injecting a NeuRet vector carrying a green fluorescent protein gene into the sub-regions of dorsal striatum in rats, followed by immunohistochemistry to detect the transgene expression in the brain.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144537798","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":"Revisiting the enchanted loom: an interpretation of reality considering epistemology.","authors":"Sén Takeda","doi":"10.1007/s12565-025-00861-2","DOIUrl":"https://doi.org/10.1007/s12565-025-00861-2","url":null,"abstract":"","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526108","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":"Visualization of nanostructures in neuronal growth cones and axons using super-resolution structured illumination microscopy.","authors":"Motohiro Nozumi, Michihiro Igarashi","doi":"10.1007/s12565-025-00863-0","DOIUrl":"https://doi.org/10.1007/s12565-025-00863-0","url":null,"abstract":"<p><p>In neurons, which possess highly organized cellular structures, such as growth cones, axons, synapses, and dendritic spines, precise determination of molecular localization is a powerful approach for elucidating cellular functions. Super-resolution microscopy has revealed higher-order structures and molecular distributions that were previously undetectable using conventional confocal microscopy. Among super-resolution techniques, structured illumination microscopy (SIM) is particularly well-suited for live-cell imaging. In this study, we present a detailed methodology for observing growth cones and axons using SIM. Our SIM imaging of primary mouse neurons revealed that the phosphorylated GAP-43 localizes within axons in a pattern consistent with the membrane-associated periodic skeleton.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526109","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":"Combination method of macroscopic imaging with MRI and microscopic imaging with super resolution microscopy for brain microstructure analysis.","authors":"Yuka Nakano, Kenji F Tanaka, Yoshifumi Abe","doi":"10.1007/s12565-025-00862-1","DOIUrl":"https://doi.org/10.1007/s12565-025-00862-1","url":null,"abstract":"<p><p>Identifying disease-relevant and therapy-related brain regions remains a major challenge in studies using animal models of psychiatric disorders. Conventional hypothesis-driven approaches often result in limited or subjective identification of brain regions. In this study, we propose an integrative method combining unbiased whole-brain structural magnetic resonance imaging (MRI) screening and detailed histological analysis. Our methodology uses structural MRI to systematically detect volumetric changes across the entire brain, enabling the identification of target regions without relying on predefined hypotheses. Once brain regions are identified, super-resolution microscopy (SRM) is employed to determine microstructural alterations at the cellular level, focusing on neurons and glial cells within those regions. To exemplify the utility of this method, we applied it to a mouse model treated with electroconvulsive therapy (ECT), an intervention which is known to increase hippocampal volume. Our demonstration highlights the potential of this approach to systematically search for brain regions of interest, providing valuable insights and guiding future studies toward a more focused exploration of key aspects of psychiatric disorder research, both in terms of pathophysiology and therapeutic action.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526106","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":"Reflections on \"Enhancing heart anatomy education in middle schools\": integrating artificial intelligence, educational equity, and instructional coherence.","authors":"Betül Digilli Ayaş","doi":"10.1007/s12565-025-00865-y","DOIUrl":"https://doi.org/10.1007/s12565-025-00865-y","url":null,"abstract":"<p><p>The integration of digital technologies into anatomy education continues to evolve, with virtual reality (VR) and mobile applications gaining traction in enhancing student engagement and conceptual understanding. Building upon the study by Koca and Çevik Özdemir (2025), which compares the effects of VR and tablet based tools on middle school students' learning of cardiac anatomy, this commentary explores additional dimensions that could further enrich the educational landscape, particularly the role of artificial intelligence (AI) in promoting instructional coherence and inclusivity. The commentary highlights a variety of AI-driven tools that have been established in the anatomy education literature, including adaptive learning platforms, large language models like ChatGPT, three-dimensional visualization environments, and digital cadaver systems such as the Anatomage Table. Moreover, the role of intelligent tutoring systems (e.g., Smart Tutor, Why2-Atlas), NLP-based platforms (e.g., IBM Watson Tutor, Ada), and AI-supported feedback systems is discussed in relation to their capacity to personalize learning experiences and enhance accessibility. Special attention is also given to pedagogical equity, addressing how AI can support students with diverse learning needs by dynamically tailoring instructional content. By synthesizing current findings and technological advancements, this commentary advocates for a more integrative approach to digital anatomy instruction, one that merges immersive technologies with responsive AI systems to foster deeper understanding, learner autonomy, and broader educational inclusion.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526107","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}
Xiaowen Wang, Marta Vittani, Ashley Bomin Lee, Philip Gade Knak, Hajime Hirase
{"title":"Genetic tools for imaging microcirculation via plasma labeling.","authors":"Xiaowen Wang, Marta Vittani, Ashley Bomin Lee, Philip Gade Knak, Hajime Hirase","doi":"10.1007/s12565-025-00858-x","DOIUrl":"https://doi.org/10.1007/s12565-025-00858-x","url":null,"abstract":"<p><p>Cerebral microcirculation is a critical infrastructure for brain function, delivering energy substrates and clearing metabolic byproducts. Disruptions in vascular dynamics contribute to neurodegenerative diseases, stroke, and cognitive impairments. Traditional blood labeling methods for fluorescence imaging, such as fluorescent dextran injection, have advanced our understanding of microcirculation but are limited for long-term imaging. In this mini review, we introduce two recently developed molecular genetic techniques, achieved by recombinant adeno-associated virus (AAV)-mediated plasma label expression or genomic knock-in that enable stable, long-term microcirculation imaging. These AAV-mediated methods require only a single systemic injection, facilitating longitudinal imaging of microcirculation in mouse models of disease. We discuss the fundamental design concepts of these approaches and explore their potential applications in systems biology.</p>","PeriodicalId":7816,"journal":{"name":"Anatomical Science International","volume":" ","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504543","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}