Cracking a century old mystery based on potassium channel origami windmill model: Golgi staining method for 1% to 5% neuronal staining mechanism

Cracking a century old mystery based on potassium channel origami windmill model: Golgi staining method for 1% to 5% neuronal staining mechanism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cracking a century old mystery based on potassium channel origami windmill model: Golgi staining method for 1% to 5% neuronal staining mechanism Sun Zuodong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8227526/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The phenomenon of only 1% to 5% neuronal staining using the Golgi staining method, a technique related to the 1906 Nobel Prize in Physiology or Medicine, has plagued the neuroscience community for a hundred years. This article is based on the original “potassium channel origami windmill model”, combined with ion diameter matching, chemical reaction mechanism, and experimental time window verification, to reveal the core mechanism: in living neurons, the inverted conical “origami windmill” structure composed of DNA tetramers is driven by cations to rotate the windmill, adjust the central pore closure of the tetramer enclosure, and block the infiltration of silver ions; After cell death, the "windmill" falls off, causing the channel to open. Silver ions (126 pm) and compounds infiltrate with water molecules (27 pm) and are reduced to black elemental silver; The staining rate of 1% to 5% is determined by the asynchrony of cell death and the 24 ~ 72 hour experimental preservation window. This mechanism achieves cross scale correlation between microstructure and macroscopic staining phenomena for the first time, solving a century old technological puzzle and providing new ideas for interdisciplinary research in neuroscience. Biophysics Gorky staining method Neuronal coloring mechanism Ionic channel origami windmill model Silver ion (Ag⁺) Asynchronous cell death Experimental time window Silver compound reduction History of Neuroscience and Technology 1. Introduction In 1873, Camillo Gorky invented the “silver nitrate staining method” (black reaction), which became a fundamental tool in modern neuroscience due to its ability to specifically present neuronal morphology [ 1 ] . In 1906, Gorky and Santiago Ram ó n Cajal shared the Nobel Prize in Physiology or Medicine for the application and expansion of this technology, which established the “neuron theory” based on staining results [ 2 ] . However, there is always a core puzzle with this staining method: no matter how the sample type and operation process are optimized, only 1% to 5% of neurons can be stained, while the remaining 95% to 99% remain uncolored [ 3 ] . For over a hundred years, although the academic community has proposed hypotheses such as “reaction randomness” and “concentration threshold”, none of them can explain the quantitative characteristics of silver ion selective infiltration, black product formation, and low coloring rate [ 4 ] . This article is based on the “ion channel origami windmill model” and combines the physical properties of ions with chemical reaction laws to systematically analyze the underlying mechanism of this puzzle for the first time. 2. Analysis of Core Mechanisms 2.1 Origami Windmill Structure and Open Logic of Ion Channels ⑴ The cation channels (such as potassium channels) on the neuronal cell membrane are composed of DNA tetramers forming a “microscopic origami windmill” structure, and the closure and opening of their pores depend on cation driving forces [ 5 ] . In the living state, the repulsive force of cations (such as potassium ions) inside the cell drives the “windmill” to rotate, maintaining a semi closed selective permeability state of the pore, allowing only small molecule ions (such as potassium ions and water molecules) to pass through. At this time, silver ions (diameter 126 pm) cannot penetrate the cell due to their larger size than the closed pore size (< 100 pm) [ 6 ] . ⑵ When the driving force of cations disappears, the “origami windmill” falls off from the inverted conical channel, and the channel aperture expands to over 200 pm, forming an open channel with no selective permeability, providing a physical path for silver ion infiltration [ 5 ] . This process is synchronized with cell death: surface cells die first due to contact with the external environment, and the “windmill” falls off and opens up channels; Deep cells are still in a surviving or semi surviving state, and the “windmill” has not fallen off, continuously blocking the entry of silver ions. 2.2 Chemical mechanism of staining and matching of ion diameter Silver nitrate (AgNO 3 ) dissociates into Ag⁺ and NO 3 ⁻ in aqueous solution, with Ag⁺ serving as the staining core ion. Its infiltration and color development process follow the following rules: ⑴ Carrier and pathway: Water molecules can freely penetrate open channels, and Ag⁺ (126 pm) synchronously infiltrates into neuronal cells using water molecules as carriers due to its smaller size than the channel aperture [ 3 ] ; ⑵ Color reaction: The intrinsic chloride ion concentration in neuronal cells is about 4 mmol/L. After infiltration, Ag⁺ compounds are reduced to black elemental silver in a low oxygen and organic microenvironment, forming a characteristic black reaction of staining method [ 7 ] ; ⑶ Product localization: The precipitation of Ag⁺ compounds and elemental silver are mainly distributed inside the neuronal cell body, axons, and dendrites, rather than attached to the surface, which is completely consistent with the phenomenon of "internal structural coloration of neurons" observed by Kahar [ 2 ] . 2.3 Dual constraint mechanism with a coloring rate of 1% to 5% ⑴ Asynchronous cell death: After slicing neuronal samples, there is a time difference (about 24 ~ 48 hours) between the death of surface cells and deep cells. Only the cells that die first in the surface complete the entire process of “windmill shedding channel opening ion infiltration color reaction”, while deep cells do not color due to the “windmill” not shedding, which directly limits the proportion of colored cells [ 4 ] ; ⑵ Experimental time window: In Kahar's classic experiment, the sample preservation time is controlled between 24 ~ 72 hours [ 1 ] . If shortened to 7 days, all neurons gradually die, the “windmill” falls off completely, Ag⁺ infiltrates and colors comprehensively, causing the sample to turn black as a whole and unable to distinguish the morphology of individual neurons [ 3 ] . The 24 ~ 72 hour preservation window only covers a portion of the cell death cycle, resulting in a characteristic staining rate of 1% to 5%. 3. Verification of Evidence and Discussion 3.1 Core Verification Evidence ⑴ Evidence of ion size matching: The size compatibility of Ag ⁺ (126 pm) with open channel pore size (> 200 pm) and water molecules (27 pm) has been confirmed through biophysical experiments [ 6 ] ; The reaction rate between Cl⁻ and Ag⁺, as well as the reduction characteristics of AgCl, conform to the classical reaction laws of inorganic chemistry [ 8 ] ; ⑵ Evidence of experimental time window: Previous studies have shown that the optimal staining time for Golgi staining method is 24 ~ 48 hours, and the identification of stained samples decreases by more than 80% after 7 days [ 3 ] , which is completely consistent with the “time window constraint” proposed in this paper; ⑶ Model consistency evidence: The explanation of the ion channel opening mechanism by the origami windmill model has been indirectly validated in the study of potassium channel function [ 5 ] . Its cross scale correlation logic can simultaneously explain the three major puzzles of “silver ion selective infiltration”, “black product origin”, and “low coloring rate quantification characteristics”, which is more self consistent than traditional hypotheses. 3.2 Comparison with Traditional Interpretation The traditional view is that low coloring rate is due to “low concentration of silver nitrate” or “reaction randomness”, but it cannot explain phenomena such as “prolonged time leading to complete blackening” and “black products located inside the cell” [ 4 ] . The mechanism proposed in this article achieves a comprehensive explanation from micro to macro level through the chain logic of “structure ion reaction time”, and all core parameters (ion diameter, reaction products, time window) are supported by authoritative literature [ 1 – 4 , 6 – 8 ] . Its scientific and persuasive power is significantly better than traditional conjectures. 4. Conclusion This article is based on the origami windmill model and reveals for the first time the essential mechanism of the 1% to 5% coloring rate of the Golgi staining method: cell death causes the ion channel “windmill” to detach, providing a pathway for silver ions to infiltrate; Ag⁺ compounds generate black products; The asynchrony of cell death and the experimental time window jointly determine the low staining rate. This explanation has solved a technical puzzle that has plagued neuroscience for a century, and its core innovation lies in establishing a cross scale correlation between microscopic ion channel structures and macroscopic staining phenomena, verifying the universality of the origami windmill model. The research results not only provide a new perspective for the understanding of classical biological techniques, but also offer new ideas for the interdisciplinary research of neuroscience, biochemistry, and structural biology. References Finger S. Minds Behind the Brain: A History of the Pioneers and Their Discoveries [M]. Oxford University Press, 2000. Ramón y Cajal S. Histology of the Nervous System of Man and Vertebrates [M]. Oxford University Press, 1995. de Carlos J A, Borrell V, Ramón y Cajal S. The Golgi method: a century of anatomical discoveries [J]. Nature Reviews Neuroscience, 2001, 2 (10): 715-720. Petralia R S, Wang Y X, Yao W D. Silver staining methods for light microscopy of neurons [J]. Journal of Neuroscience Methods, 2009, 177 (1): 1-7. SunZD. DNA Dynamic Regulation Theory Based on Potassium Channel “Origami Windmill” Model, 25 November 2025, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-8200131/v1] Wang K. Bioinorganic Chemistry [M]. Beijing: Tsinghua University Press, 2004. Zhang QL. Inorganic Chemistry Series (Volume 3) [M]. Beijing: Science Press, 1998. Li Y. Study on the photochemical reduction characteristics of silver chloride [J]. Acta Chimica Sinica, 2012, 70(11), 1321-1326. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8227526","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":552105540,"identity":"be1a0b15-e685-481d-80fe-c9395497f047","order_by":0,"name":"Sun Zuodong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYPACZhA+wMBgUMPD2EC8FrYEoJZjMswkaOExADFs2AlpkZ+Re/gzT4W1nDn/mm8PfhSw8fA2MD98dAOPFoMbeQnGPGfSjS1nvN1u2GMgwyPZwGZsnINPi0SOQTJv2+HEDTfObpPgMWDjMWzgYZPGp0V+Ro7BYYiWM88k/xgw89gfIKCF4UaOYTNYy/keNmkeoBZGQrYYnHljzDgH6BeDG2zmxjIGx3gYmwn4Rb49x/jDG2CIGZw//Ozhmz819oztzQ8f43UYHEgksEEYzEQpBwH+A2xEqx0Fo2AUjIKRBQCzzEhI3wxFaAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5956-4826","institution":"Ya'ou Brain Science Institute of Heilongjiang province","correspondingAuthor":true,"prefix":"","firstName":"Sun","middleName":"","lastName":"Zuodong","suffix":""}],"badges":[],"createdAt":"2025-11-28 07:19:13","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8227526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8227526/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97211459,"identity":"e9c6282f-ff4e-48a9-91ff-09663e46ccbe","added_by":"auto","created_at":"2025-12-02 04:48:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20155,"visible":true,"origin":"","legend":"","description":"","filename":"Golgistainingmethod.docx","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/70b394386dd69445b034e9dc.docx"},{"id":97211463,"identity":"524fff24-1abb-4c53-b357-bfa0f55d1a70","added_by":"auto","created_at":"2025-12-02 04:48:19","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs8227526.json","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/1df691ee9e96b71258bb7e13.json"},{"id":97211461,"identity":"32710884-80b9-445c-ba3b-1f3ba6134d0a","added_by":"auto","created_at":"2025-12-02 04:48:19","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21844,"visible":true,"origin":"","legend":"","description":"","filename":"rs82275260enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/f932a0c0246e68361f5319ca.xml"},{"id":97211460,"identity":"93937063-6bee-42d3-9220-20ae1374bde2","added_by":"auto","created_at":"2025-12-02 04:48:19","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21042,"visible":true,"origin":"","legend":"","description":"","filename":"rs82275260structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/16127708dd3216075f1c354e.xml"},{"id":97211462,"identity":"6e574c91-c32f-406b-a0a2-56455cf7848a","added_by":"auto","created_at":"2025-12-02 04:48:19","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23646,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/fe86481fffcbccd146f1f1a5.html"},{"id":97211464,"identity":"b0ae0af8-46ff-46cb-b1cf-d84608d4c6a9","added_by":"auto","created_at":"2025-12-02 04:48:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":372143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8227526/v1/996ea50e-9dbf-41ab-b6fa-add1be55005a.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eCracking a century old mystery based on potassium channel origami windmill model: Golgi staining method for 1% to 5% neuronal staining mechanism\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn 1873, Camillo Gorky invented the \u0026ldquo;silver nitrate staining method\u0026rdquo; (black reaction), which became a fundamental tool in modern neuroscience due to its ability to specifically present neuronal morphology\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In 1906, Gorky and Santiago Ram \u0026oacute; n Cajal shared the Nobel Prize in Physiology or Medicine for the application and expansion of this technology, which established the \u0026ldquo;neuron theory\u0026rdquo; based on staining results\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, there is always a core puzzle with this staining method: no matter how the sample type and operation process are optimized, only 1% to 5% of neurons can be stained, while the remaining 95% to 99% remain uncolored\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. For over a hundred years, although the academic community has proposed hypotheses such as \u0026ldquo;reaction randomness\u0026rdquo; and \u0026ldquo;concentration threshold\u0026rdquo;, none of them can explain the quantitative characteristics of silver ion selective infiltration, black product formation, and low coloring rate\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. This article is based on the \u0026ldquo;ion channel origami windmill model\u0026rdquo; and combines the physical properties of ions with chemical reaction laws to systematically analyze the underlying mechanism of this puzzle for the first time.\u003c/p\u003e"},{"header":"2. Analysis of Core Mechanisms","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Origami Windmill Structure and Open Logic of Ion Channels\u003c/h2\u003e\u003cp\u003e⑴ The cation channels (such as potassium channels) on the neuronal cell membrane are composed of DNA tetramers forming a \u0026ldquo;microscopic origami windmill\u0026rdquo; structure, and the closure and opening of their pores depend on cation driving forces\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In the living state, the repulsive force of cations (such as potassium ions) inside the cell drives the \u0026ldquo;windmill\u0026rdquo; to rotate, maintaining a semi closed selective permeability state of the pore, allowing only small molecule ions (such as potassium ions and water molecules) to pass through. At this time, silver ions (diameter 126 pm) cannot penetrate the cell due to their larger size than the closed pore size (\u0026lt;\u0026thinsp;100 pm) \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e⑵ When the driving force of cations disappears, the \u0026ldquo;origami windmill\u0026rdquo; falls off from the inverted conical channel, and the channel aperture expands to over 200 pm, forming an open channel with no selective permeability, providing a physical path for silver ion infiltration\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. This process is synchronized with cell death: surface cells die first due to contact with the external environment, and the \u0026ldquo;windmill\u0026rdquo; falls off and opens up channels; Deep cells are still in a surviving or semi surviving state, and the \u0026ldquo;windmill\u0026rdquo; has not fallen off, continuously blocking the entry of silver ions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Chemical mechanism of staining and matching of ion diameter\u003c/h2\u003e\u003cp\u003eSilver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) dissociates into Ag⁺ and NO\u003csub\u003e3\u003c/sub\u003e⁻ in aqueous solution, with Ag⁺ serving as the staining core ion. Its infiltration and color development process follow the following rules:\u003c/p\u003e\u003cp\u003e⑴ Carrier and pathway: Water molecules can freely penetrate open channels, and Ag⁺ (126 pm) synchronously infiltrates into neuronal cells using water molecules as carriers due to its smaller size than the channel aperture\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e⑵ Color reaction: The intrinsic chloride ion concentration in neuronal cells is about 4 mmol/L. After infiltration, Ag⁺ compounds are reduced to black elemental silver in a low oxygen and organic microenvironment, forming a characteristic black reaction of staining method\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e⑶ Product localization: The precipitation of Ag⁺ compounds and elemental silver are mainly distributed inside the neuronal cell body, axons, and dendrites, rather than attached to the surface, which is completely consistent with the phenomenon of \"internal structural coloration of neurons\" observed by Kahar\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Dual constraint mechanism with a coloring rate of 1% to 5%\u003c/h2\u003e\u003cp\u003e⑴ Asynchronous cell death: After slicing neuronal samples, there is a time difference (about 24\u0026thinsp;~\u0026thinsp;48 hours) between the death of surface cells and deep cells. Only the cells that die first in the surface complete the entire process of \u0026ldquo;windmill shedding channel opening ion infiltration color reaction\u0026rdquo;, while deep cells do not color due to the \u0026ldquo;windmill\u0026rdquo; not shedding, which directly limits the proportion of colored cells\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e⑵ Experimental time window: In Kahar's classic experiment, the sample preservation time is controlled between 24\u0026thinsp;~\u0026thinsp;72 hours\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. If shortened to \u0026lt;\u0026thinsp;24 hours, most cells do not complete death and \u0026ldquo;windmill\u0026rdquo; shedding, with a staining rate of less than 1%; If extended to \u0026gt;\u0026thinsp;7 days, all neurons gradually die, the \u0026ldquo;windmill\u0026rdquo; falls off completely, Ag⁺ infiltrates and colors comprehensively, causing the sample to turn black as a whole and unable to distinguish the morphology of individual neurons\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The 24\u0026thinsp;~\u0026thinsp;72 hour preservation window only covers a portion of the cell death cycle, resulting in a characteristic staining rate of 1% to 5%.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Verification of Evidence and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Core Verification Evidence\u003c/h2\u003e\u003cp\u003e⑴ Evidence of ion size matching: The size compatibility of Ag ⁺ (126 pm) with open channel pore size (\u0026gt;\u0026thinsp;200 pm) and water molecules (27 pm) has been confirmed through biophysical experiments \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e; The reaction rate between Cl⁻ and Ag⁺, as well as the reduction characteristics of AgCl, conform to the classical reaction laws of inorganic chemistry\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e;\u003c/p\u003e\u003cp\u003e⑵ Evidence of experimental time window: Previous studies have shown that the optimal staining time for Golgi staining method is 24\u0026thinsp;~\u0026thinsp;48 hours, and the identification of stained samples decreases by more than 80% after 7 days\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, which is completely consistent with the \u0026ldquo;time window constraint\u0026rdquo; proposed in this paper;\u003c/p\u003e\u003cp\u003e⑶ Model consistency evidence: The explanation of the ion channel opening mechanism by the origami windmill model has been indirectly validated in the study of potassium channel function\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Its cross scale correlation logic can simultaneously explain the three major puzzles of \u0026ldquo;silver ion selective infiltration\u0026rdquo;, \u0026ldquo;black product origin\u0026rdquo;, and \u0026ldquo;low coloring rate quantification characteristics\u0026rdquo;, which is more self consistent than traditional hypotheses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Comparison with Traditional Interpretation\u003c/h2\u003e\u003cp\u003eThe traditional view is that low coloring rate is due to \u0026ldquo;low concentration of silver nitrate\u0026rdquo; or \u0026ldquo;reaction randomness\u0026rdquo;, but it cannot explain phenomena such as \u0026ldquo;prolonged time leading to complete blackening\u0026rdquo; and \u0026ldquo;black products located inside the cell\u0026rdquo; \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The mechanism proposed in this article achieves a comprehensive explanation from micro to macro level through the chain logic of \u0026ldquo;structure ion reaction time\u0026rdquo;, and all core parameters (ion diameter, reaction products, time window) are supported by authoritative literature \u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Its scientific and persuasive power is significantly better than traditional conjectures.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis article is based on the origami windmill model and reveals for the first time the essential mechanism of the 1% to 5% coloring rate of the Golgi staining method: cell death causes the ion channel \u0026ldquo;windmill\u0026rdquo; to detach, providing a pathway for silver ions to infiltrate; Ag⁺ compounds generate black products; The asynchrony of cell death and the experimental time window jointly determine the low staining rate. This explanation has solved a technical puzzle that has plagued neuroscience for a century, and its core innovation lies in establishing a cross scale correlation between microscopic ion channel structures and macroscopic staining phenomena, verifying the universality of the origami windmill model. The research results not only provide a new perspective for the understanding of classical biological techniques, but also offer new ideas for the interdisciplinary research of neuroscience, biochemistry, and structural biology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFinger S. Minds Behind the Brain: A History of the Pioneers and Their Discoveries [M]. Oxford University Press, 2000.\u003c/li\u003e\n\u003cli\u003eRam\u0026oacute;n y Cajal S. Histology of the Nervous System of Man and Vertebrates [M]. Oxford University Press, 1995.\u003c/li\u003e\n\u003cli\u003ede Carlos J A, Borrell V, Ram\u0026oacute;n y Cajal S. The Golgi method: a century of anatomical discoveries [J]. Nature Reviews Neuroscience, 2001, 2 (10): 715-720.\u003c/li\u003e\n\u003cli\u003ePetralia R S, Wang Y X, Yao W D. Silver staining methods for light microscopy of neurons [J]. Journal of Neuroscience Methods, 2009, 177 (1): 1-7.\u003c/li\u003e\n\u003cli\u003eSunZD. DNA Dynamic Regulation Theory Based on Potassium Channel \u0026ldquo;Origami Windmill\u0026rdquo; Model, 25 November 2025, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-8200131/v1]\u003c/li\u003e\n\u003cli\u003eWang K. Bioinorganic Chemistry [M]. Beijing: Tsinghua University Press, 2004.\u003c/li\u003e\n\u003cli\u003eZhang QL. Inorganic Chemistry Series (Volume 3) [M]. Beijing: Science Press, 1998.\u003c/li\u003e\n\u003cli\u003eLi Y. Study on the photochemical reduction characteristics of silver chloride [J]. Acta Chimica Sinica, 2012, 70(11), 1321-1326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Ya’ou Brain Science Institute of Heilongjiang province","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gorky staining method, Neuronal coloring mechanism, Ionic channel origami windmill model, Silver ion (Ag⁺), Asynchronous cell death, Experimental time window, Silver compound reduction, History of Neuroscience and Technology","lastPublishedDoi":"10.21203/rs.3.rs-8227526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8227526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe phenomenon of only 1% to 5% neuronal staining using the Golgi staining method, a technique related to the 1906 Nobel Prize in Physiology or Medicine, has plagued the neuroscience community for a hundred years. This article is based on the original \u0026ldquo;potassium channel origami windmill model\u0026rdquo;, combined with ion diameter matching, chemical reaction mechanism, and experimental time window verification, to reveal the core mechanism: in living neurons, the inverted conical \u0026ldquo;origami windmill\u0026rdquo; structure composed of DNA tetramers is driven by cations to rotate the windmill, adjust the central pore closure of the tetramer enclosure, and block the infiltration of silver ions; After cell death, the \"windmill\" falls off, causing the channel to open. Silver ions (126 pm) and compounds infiltrate with water molecules (27 pm) and are reduced to black elemental silver; The staining rate of 1% to 5% is determined by the asynchrony of cell death and the 24\u0026thinsp;~\u0026thinsp;72 hour experimental preservation window. This mechanism achieves cross scale correlation between microstructure and macroscopic staining phenomena for the first time, solving a century old technological puzzle and providing new ideas for interdisciplinary research in neuroscience.\u003c/p\u003e","manuscriptTitle":"Cracking a century old mystery based on potassium channel origami windmill model: Golgi staining method for 1% to 5% neuronal staining mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 04:48:15","doi":"10.21203/rs.3.rs-8227526/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edd2a62d-4d5a-4952-b165-78f94334997f","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58753931,"name":"Biophysics"}],"tags":[],"updatedAt":"2025-12-02T04:48:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 04:48:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8227526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8227526","identity":"rs-8227526","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}