Energetic diversity in retinal ganglion cells is modulated by neuronal activity and correlates with resilience to degeneration

Energetic diversity in retinal ganglion cells is modulated by neuronal activity and correlates with resilience to degeneration | 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 Article Energetic diversity in retinal ganglion cells is modulated by neuronal activity and correlates with resilience to degeneration Philip Williams, Zelun Wang, Christopher Zhao, Shelly Xu, Sean McCracken, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5989609/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Neuronal function requires high energy expenditure that is likely customized to meet specific signaling demands. However, little is known about diversity of metabolic homeostasis among divergently-functioning types of neurons. To this end, we examined retinal ganglion cells (RGCs), a population of closely related, yet electrophysiologically distinct excitatory projection neurons. Using in vivo 2-photon imaging to measure ATP with single cell resolution, we identified differential homeostatic energy maintenance in the RGC population that correspond to distinct RGC types. In the presence of circuit activity, the most active RGC type (Alpha RGCs), had lower homeostatic ATP levels than other types and exhibited the greatest magnitude of ATP decline when ATP synthesis was inhibited. By simultaneously manipulating circuit activity and mitochondrial function, we found that while oxidative phosphorylation was required to meet ATP demands during circuit activity, it was expendable to maintain resting ATP levels. We also examined ATP signatures associated with survival and injury response after axotomy and report a correlation between low homeostatic ATP and increased survival. In addition, we observed transient ATP increases in RGCs following axon injury. Together, these findings identify diversity of energy handling capabilities of dynamically active neurons with implications for neuronal resilience. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Neuroscience/Stress and resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations There is NO Competing Interest. Key Resource Table The Key Resource Table file is not available with this version. Supplementary Files WangetalFigureS1TOMsm.pdf Supplementary Figure 1. Mitochondrial protein expression is higher in aRGCs. (A) Confocal maximum intensity projections (mip) of retina wholemounts immunostained for RBPMS (magenta), SPP1 (cyan) and the indicated mitochondrial component (yellow). Cyan arrows indicate SPP1 + αRGCs, magenta arrows indicate SPP1 - RGCs. Scale bars = 50 μm. (B) Boxplots of relative mitochondrial component expression intensity in SPP1 - (magenta) and SPP1 + (cyan) RGCs (TOM20 n = 2218 RGCs from 3 retinas, NDUFB8 n = 2204 RGCs from 3 retinas, SDHA n = 2604 RGCs from 4 retinas, UQCRC n = 2063 RGCs from 3 retinas, ATP5A n= 2170 RGCs from 3 retinas). ** p < 0.001, Student’s t-test. WangetalFigureS2sm.pdf Supplementary Figure 2. Activity modulation induces Ca2+ changes in RGCs in vivo . (A) In vivo 2-photon aips of Twitch2b in the same retina imaged before and at the indicated time points after intravitreal injection of Str/Bic. YFP and CFP pseuodocolored red and cyan respectively throughout. (B) Traces of single RGC Ca2+ levels tracked in an example retina in response to Str/Bic. Each line indicates an individual RGC. zScores are pseudocolored according to the values in the pre trace. (C) Scatter and box plots of RGC Ca2+ levels before and at the indicated time points after intravitreal injection of the Str/Bic. Each point represents a single RGC. Colors indicates time points as represented by the boxed regions in (B). ANOVA with posthoc Tukey’s test. (D-E) Scatterplots of Ca2+ levels before and at the indicated time points after intravitreal injection of the Str/Bic (n=923 RGCs from 6 retinas). (F) In vivo 2-photon aips of the same retina imaged before and at the indicated time points after intravitreal injection of NBQX/AP5. Magenta arrows indicate RGCs with increased Ca2+ after injection. (G) Traces of single RGC Ca2+ levels tracked in an example retina in response to NBQX/AP5. (H) Scatter and box plots of RGC Ca2+ levels before and at the indicated time points after intravitreal injection of the NBQX/AP5. (I-J) Scatterplots of Ca2+ levels before and at the indicated time points after intravitreal injection of the NBQX/AP5 (n=334 RGCs from 3 retinas). (K) In vivo 2-photon aips of a retina dark adapted for 2 minutes and then imaged immediately after scanning onset (left) or 4 minutes later after reaching steady state (right). (L) Traces of single RGC Ca2+ levels tracked in a dark adapted example retina from the time point immediately after scanning onset. (M) Scatterpot of the change in Ca2+ during the timeframe immediately after scanning onset or after reaching steady state indicated by the purple and orange boxes in (L) respectively. Kruskal-Wallis test with posthoc Dunn’s test. (n=2055 RGCs from 13 retinas). Scale bars = 50 μm. 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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-5989609","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":424153776,"identity":"08c6dd01-24c5-466e-ab60-a41bdfd625eb","order_by":0,"name":"Philip Williams","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYJCCAw8KJBj4wcwCEJcYLQkGEgySDSCWAZFaGBKAKg0OEKvFXPqMIdAWC3njGznGnz8YMMjx3UjAr8WyL8cA5DDDbTdyzCSAthhLEtJicIZ3A0gL47bbOWYghyVuIFaL/ebZOcYfgFrqidaSuEE6xwDksAQDgn7p4f8A0pI84/6zMokzQE/NPPMAvxZzHrbkDx8q6mz7ew5v/lBRYSPPd5yQw9D4EviVY9MyCkbBKBgFowATAACVVUgvKrl5ggAAAABJRU5ErkJggg==","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":true,"prefix":"","firstName":"Philip","middleName":"","lastName":"Williams","suffix":""},{"id":424153778,"identity":"f3f5366b-0660-4f15-97cf-693389e044ab","order_by":1,"name":"Zelun Wang","email":"","orcid":"https://orcid.org/0000-0003-3768-6934","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Zelun","middleName":"","lastName":"Wang","suffix":""},{"id":424153780,"identity":"dfb8076c-14bc-4b13-834f-877cf5fd03a7","order_by":2,"name":"Christopher Zhao","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Zhao","suffix":""},{"id":424153782,"identity":"c3673afe-f59f-4007-b55d-5a2d1a9744e2","order_by":3,"name":"Shelly Xu","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Shelly","middleName":"","lastName":"Xu","suffix":""},{"id":424153784,"identity":"a41cb39e-3af3-47cb-9650-25f4581263a8","order_by":4,"name":"Sean McCracken","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Sean","middleName":"","lastName":"McCracken","suffix":""},{"id":424153786,"identity":"1146b0bf-e52e-4e48-b24b-52fae95aa3f7","order_by":5,"name":"Rajendra Apte","email":"","orcid":"https://orcid.org/0000-0003-2281-2336","institution":"Washington University in St. Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rajendra","middleName":"","lastName":"Apte","suffix":""}],"badges":[],"createdAt":"2025-02-08 19:55:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5989609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5989609/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78329275,"identity":"869c2973-2d4a-4cf3-8e88-fbe9234f74f5","added_by":"auto","created_at":"2025-03-12 06:53:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1077627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATP heterogeneity across RGCs. \u003c/strong\u003e(A) ATeam was expressed in RGCs by intraocular injection of a Cre-dependent AAV expression vector into VGlut2-Cre mice. Transpupillary \u003cem\u003ein vivo \u003c/em\u003e2-photon imaging measured Cp173-mVenus (YFP) and mseCFP (CFP) intensity of ATeam in RGC somas segmented with Cellpose. (B) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon average intensity projection (aip) of ATeam time lapse image series. YFP and CFP pseuodocolored magenta and green respectively throughout. Arrows indicate RGCs with lower (green) and higher (magenta) ATP levels. (C) Traces of single RGC ATP levels tracked in an example retina, 20 frame moving average graphed throughout. zScores are pseudocolored from minimal (blue) to maximal (yellow) by average over the sample trace. (D) Histogram of ATP level distributions across multiple retinas (n=537 RGCs from 5 retinas). Each retina is represented by a unique color. (E) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of the same retina imaged 7 days apart. Arrows indicate the same example RGCs. (F) Scatterplot of ATP levels measured in RGCs initially and 7 days later. Each point represents a single RGC. R = Pearson’s correlation coefficient throughout (n=381 RGCs from 7 retinas). (G) \u003cem\u003eIn vivo \u003c/em\u003e2-photon aip of ATeam (upper left). Confocal maximum intensity projection (mip) of fixed YFP signal from ATeam (lower left), and indicated RGC type marker immunostaining for CART (yellow), TBR2 (magenta) and SPP1 (cyan). Arrows indicate example RGCs and match type coloring on left panels. (H) Scatter and boxplots of ATP levels by RGC types. ANOVA with posthoc Tukey’s test (n=349 RGCs from 5 retinas). Scale bars = 50 μm. Diagrams created with BioRender.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/1708ece5e2b9e868ce7e9252.png"},{"id":78329745,"identity":"bcadf637-84f6-4dc0-80fa-ce09625e946a","added_by":"auto","created_at":"2025-03-12 07:01:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":995410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRGCs are differentially affected by mitochondrial inhibition. \u003c/strong\u003e(A) Schematic of ETC inhibitors and their targets. (B-E). \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of ATeam in the same retina imaged before and after intravitreal injection of the indicated pharmacological inhibitor. Scale bars = 50 μm. (F-I). Traces of single RGC ATP levels tracked in an example retina in response to the indicated ETC inhibitor. Each line indicates an individual RGC. zScores are pseudocolored from minimal (blue) to maximal (yellow) according to the values in the pre trace. (J-M) Scatterplot of ATP levels in RGCs comparing pre and post injection of the indicated ETC inhibitor. Each point represents an RGC, and each color indicates a retina (ROT n=264 RGCs from 5 retinas, TTFA n = 212 RGCs from 4 retinas, AA n=220 RGCs from 4 retinas, KCN n=218 RGCs from 4 retinas). (N-Q) Example traces of ATP levels in (F-I) pseudocolored by RGC type according to \u003cem\u003epost hoc\u003c/em\u003e immunostaining as performed in Figure 1F. Yellow = CART\u003csup\u003e+\u003c/sup\u003e, magenta = TBR2\u003csup\u003e+\u003c/sup\u003e, cyan = SPP1\u003csup\u003e+\u003c/sup\u003e, green = TBR2\u003csup\u003e+\u003c/sup\u003eSPP1\u003csup\u003e+\u003c/sup\u003e and gray indicates negative for all markers. (R-U) Scatter and boxplots of post injection changes in ATP levels from pre (blue box in N-Q) for indicated ETC inhibitor (purple box in N-Q). ANOVA with posthoc Tukey’s test.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/199e87420aafe80dea2acb10.png"},{"id":78329278,"identity":"09306ad4-93b9-4080-a73f-2c437eff48df","added_by":"auto","created_at":"2025-03-12 06:53:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1262760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of activity modulation on ATP in RGCs. \u003c/strong\u003e(A) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of ATeam in the same retina imaged before and at the indicated time points after intravitreal injection of Str/Bic. (B) Traces of single RGC ATP levels tracked in an example retina in response to Str/Bic. Each line indicates an individual RGC. zScores are pseudocolored according to the values in the pre trace. (C) Scatter and box plots of RGC ATP levels before and at the indicated time points after intravitreal injection of the Str/Bic. Each point represents a single RGC. Colors indicate time points as represented by the boxed regions in (B). ANOVA with posthoc Tukey’s test throughout. (D-E) Scatterplots of ATP levels before and at the indicated time points after intravitreal injection of the Str/Bic (n=251 RGCs from 3 retinas). (F) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of the same retina imaged before and at the indicated time points after intravitreal injection of NBQX/AP5. (G) Traces of single RGC ATP levels tracked in an example retina in response to NBQX/AP5. (H) Scatter and box plots of RGC ATP levels before and at the indicated time points after intravitreal injection of the NBQX/AP5. (I-J) Scatterplots of ATP levels before and at the indicated time points after intravitreal injection of the NBQX/AP5 (n=282 RGCs from 4 retinas). (K)\u003cem\u003e In vivo\u003c/em\u003e 2-photon aips of a retina dark adapted for 2 minutes and then imaged immediately after scanning onset (left) or 4 minutes later after reaching steady state (right). (L) Traces of single RGC ATP levels tracked in a dark-adapted example retina from the time point immediately after scanning onset. (M) Scatterpot of the change in ATP during the timeframe immediately after scanning onset or after reaching steady state indicated by the purple and orange boxes in (L) respectively (n=485 RGCs from 4 retinas). Kruskal-Wallis test with posthoc Dunn’s test. Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/177cd879895396fe5b66f164.png"},{"id":78329277,"identity":"662b6121-888e-49ac-a340-9b95d8dbb9c1","added_by":"auto","created_at":"2025-03-12 06:53:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1011111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of activity on RGC ATP during mitochondrial inhibition. \u003c/strong\u003e(A) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of ATeam in the same retina imaged before and afterintravitreal injection of ROT. (B) Traces of single RGC ATP levels tracked in an example retina in response to ROT. Each line indicates an individual RGC. zScores are pseudocolored according to the values in the pre trace. (C)\u003cem\u003e In vivo\u003c/em\u003e 2-photon aips of the same retina imaged before and after intravitreal injection of ROT \u0026amp; NBQX/AP5. (D) Traces of single RGC ATP levels tracked in an example retina in response to ROT \u0026amp; NBQX/AP5. (E) Scatter and boxplots of changes in ATP induced by ROT alone or ROT \u0026amp; NBQX/AP5. Orange and magenta regions in (B) and (D) respectively indicate time windows compared to pre (blue) to calculate DATP. Kruskal-Wallis test with posthoc Dunn’s test (ROT n=733 RGCs from 6 retinas (data replotted from Figure 2R), ROT \u0026amp; NBQX/AP5 n=349 RGCs from 4 retinas, rank sum test). (F) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of the same retina imaged before and after intravitreal injection of ROT with a period of dark adaption during the image acquisition. (G) Traces of single RGC ATP levels tracked in an example retina in response to ROT. Solid gray bar indicates dark adaptation period. (H) Zoom in of the example traces tracked in the boxed region of (G). (I) Scatter and box plots of individual RGC ATP in response to ROT injection and a period of dark adaptation. Colors match time windows indicated in (G), blue = pre, purple = post ROT trough, red = immediately after scanning restart, orange = trough after restart. Each point is an RGC and lines connect individual RGCs over time points. ANOVA with posthoc Tukey’s test (n=284 RGCs from 3 retinas). (J) Scatter and box plots of the change in ATP during the indicated time window calculated by subtracting the minimal observed level from the maximal observed level recorded within that time window. Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/aefe6a8c10d0200d19e09a10.png"},{"id":78328293,"identity":"f952dcb4-64f9-414b-b05c-5fe8e58f65e9","added_by":"auto","created_at":"2025-03-12 06:45:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":352721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between RGC ATP and injury response. \u003c/strong\u003e(A) Timeline of optic nerve crush survival experiments. (B) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of ATeam in the same retina imaged before and at the indicated time points after optic nerve crush. Cyan arrows indicate surviving SPP1\u003csup\u003e+\u003c/sup\u003e RGCs, orange arrows indicate surviving SPP1\u003csup\u003e-\u003c/sup\u003e RGCs, magenta to red arrows indicate dying RGCs. Scale bars = 50 μm. (C) Scatter and boxplots of homeostatic ATP levels in the pre-injury time point for RGCs that eventually died or survived the 14-day time lapse. Each point indicates an individual RGC (n=264 RGCs from 5 retinas, paired t-test). (D) Scatter and boxplots of dying RGCs with the surviving aRGCs identified by \u003cem\u003epost hoc\u003c/em\u003e immunostaining of SPP1 separated out from the surviving cohort (n=264 RGCs from 5 retinas, One-way ANOVA with Tukey’s correction). (E) Line plot of ATP dynamics after optic nerve crush separated by dying (blue) and surviving (orange) RGCs. Lines represent mean, shaded area represents +/- sd, points represent retina sample means (n=188 RGCs from 3 retinas, ranked sum test of all RGCs at time point vs. pre).\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/e1c03fd37daa674b9e7728ed.png"},{"id":78331293,"identity":"2b16b87f-6d33-40d4-9e5f-da7aad6881cd","added_by":"auto","created_at":"2025-03-12 07:09:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1093116,"visible":true,"origin":"","legend":"","description":"","filename":"WangFullmanuscript020825.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1_covered_dade4a74-cf9e-4232-8940-95f5c79d14aa.pdf"},{"id":78328290,"identity":"204826cf-e13a-4a65-b604-3fe8fe2c21ce","added_by":"auto","created_at":"2025-03-12 06:45:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3015843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Mitochondrial protein expression is higher in aRGCs. \u003c/strong\u003e(A) Confocal maximum intensity projections (mip) of retina wholemounts immunostained for RBPMS (magenta), SPP1 (cyan) and the indicated mitochondrial component (yellow). Cyan arrows indicate SPP1\u003csup\u003e+\u003c/sup\u003e αRGCs, magenta arrows indicate SPP1\u003csup\u003e-\u003c/sup\u003e RGCs. Scale bars = 50 μm. (B) Boxplots of relative mitochondrial component expression intensity in SPP1\u003csup\u003e-\u003c/sup\u003e (magenta) and SPP1\u003csup\u003e+\u003c/sup\u003e (cyan) RGCs (TOM20 n = 2218 RGCs from 3 retinas, NDUFB8 n = 2204 RGCs from 3 retinas, SDHA n = 2604 RGCs from 4 retinas, UQCRC n = 2063 RGCs from 3 retinas, ATP5A n= 2170 RGCs from 3 retinas). ** p \u0026lt; 0.001, Student’s t-test.\u003c/p\u003e","description":"","filename":"WangetalFigureS1TOMsm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/c891f681d6537e057f9d62ad.pdf"},{"id":78328285,"identity":"5a23cda4-daa4-4e97-b9a7-6f039ac70764","added_by":"auto","created_at":"2025-03-12 06:45:49","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1886007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Activity modulation induces Ca2+ changes in RGCs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) \u003cem\u003eIn vivo\u003c/em\u003e2-photon aips of Twitch2b in the same retina imaged before and at the indicated time points after intravitreal injection of Str/Bic. YFP and CFP pseuodocolored red and cyan respectively throughout. (B) Traces of single RGC Ca2+ levels tracked in an example retina in response to Str/Bic. Each line indicates an individual RGC. zScores are pseudocolored according to the values in the pre trace. (C) Scatter and box plots of RGC Ca2+ levels before and at the indicated time points after intravitreal injection of the Str/Bic. Each point represents a single RGC. Colors indicates time points as represented by the boxed regions in (B). ANOVA with posthoc Tukey’s test. (D-E) Scatterplots of Ca2+ levels before and at the indicated time points after intravitreal injection of the Str/Bic (n=923 RGCs from 6 retinas). (F) \u003cem\u003eIn vivo\u003c/em\u003e 2-photon aips of the same retina imaged before and at the indicated time points after intravitreal injection of NBQX/AP5. Magenta arrows indicate RGCs with increased Ca2+ after injection. (G) Traces of single RGC Ca2+ levels tracked in an example retina in response to NBQX/AP5. (H) Scatter and box plots of RGC Ca2+ levels before and at the indicated time points after intravitreal injection of the NBQX/AP5. (I-J) Scatterplots of Ca2+ levels before and at the indicated time points after intravitreal injection of the NBQX/AP5 (n=334 RGCs from 3 retinas). (K)\u003cem\u003e In vivo\u003c/em\u003e 2-photon aips of a retina dark adapted for 2 minutes and then imaged immediately after scanning onset (left) or 4 minutes later after reaching steady state (right). (L) Traces of single RGC Ca2+ levels tracked in a dark adapted example retina from the time point immediately after scanning onset. (M) Scatterpot of the change in Ca2+ during the timeframe immediately after scanning onset or after reaching steady state indicated by the purple and orange boxes in (L) respectively. Kruskal-Wallis test with posthoc Dunn’s test. (n=2055 RGCs from 13 retinas). Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"WangetalFigureS2sm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989609/v1/49aebd756af8855aeb88e017.pdf"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e\n\u003cp\u003eKey Resource Table\u003c/p\u003e\n\u003cp\u003eThe Key Resource Table file is not available with this version.\u003c/p\u003e","formattedTitle":"Energetic diversity in retinal ganglion cells is modulated by neuronal activity and correlates with resilience to degeneration","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5989609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5989609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuronal function requires high energy expenditure that is likely customized to meet specific signaling demands. However, little is known about diversity of metabolic homeostasis among divergently-functioning types of neurons. To this end, we examined retinal ganglion cells (RGCs), a population of closely related, yet electrophysiologically distinct excitatory projection neurons. Using \u003cem\u003ein vivo\u003c/em\u003e 2-photon imaging to measure ATP with single cell resolution, we identified differential homeostatic energy maintenance in the RGC population that correspond to distinct RGC types. In the presence of circuit activity, the most active RGC type (Alpha RGCs), had lower homeostatic ATP levels than other types and exhibited the greatest magnitude of ATP decline when ATP synthesis was inhibited. By simultaneously manipulating circuit activity and mitochondrial function, we found that while oxidative phosphorylation was required to meet ATP demands during circuit activity, it was expendable to maintain resting ATP levels. We also examined ATP signatures associated with survival and injury response after axotomy and report a correlation between low homeostatic ATP and increased survival. In addition, we observed transient ATP increases in RGCs following axon injury. Together, these findings identify diversity of energy handling capabilities of dynamically active neurons with implications for neuronal resilience.\u003c/p\u003e","manuscriptTitle":"Energetic diversity in retinal ganglion cells is modulated by neuronal activity and correlates with resilience to degeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 06:45:44","doi":"10.21203/rs.3.rs-5989609/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec97d383-9aeb-4a2b-a326-7163d0137398","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":45201187,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":45201190,"name":"Biological sciences/Neuroscience/Stress and resilience"}],"tags":[],"updatedAt":"2026-03-10T11:31:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 06:45:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5989609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5989609","identity":"rs-5989609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}