{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T03:10:22Z","timestamp":1792465822181,"version":"4.5.0"},"reference-count":39,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T00:00:00Z","timestamp":1667347200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003693","name":"Korea Institute of Science and Technology","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003693","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008907","name":"Oklahoma Medical Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008907","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001298","name":"Presbyterian Health Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100001298","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Neurosci."],"abstract":"<jats:p>\n                    Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer\u2019s disease and Parkinson\u2019s disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    . This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril \u03b1-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, \u03b1-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.\n                  <\/jats:p>","DOI":"10.3389\/fnmol.2022.1042616","type":"journal-article","created":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T05:15:40Z","timestamp":1667366140000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":23,"title":["Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion"],"prefix":"10.3389","volume":"15","author":[{"given":"Su Yeon","family":"Kim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaudia","family":"Strucinska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bertha","family":"Osei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kihoon","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seok-Kyu","family":"Kwon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr.","given":"Tommy L.","family":"Lewis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,11,2]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1159\/000454924","article-title":"Energy metabolism and ageing in the mouse: A mini-review.","volume":"63","author":"Azzu","year":"2017","journal-title":"Gerontology"},{"key":"B2","doi-asserted-by":"publisher","first-page":"724","DOI":"10.1016\/j.cmet.2011.08.016","article-title":"Brain energy metabolism: Focus on astrocyte-neuron metabolic cooperation.","volume":"14","author":"Belanger","year":"2011","journal-title":"Cell Metab."},{"key":"B3","doi-asserted-by":"publisher","first-page":"289","DOI":"10.30802\/AALAS-JAALAS-20-000143","article-title":"Effects of oxygen supplementation on injectable and inhalant anesthesia in C57BL\/6 mice.","volume":"60","author":"Blevins","year":"2021","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"B4","author":"Buffalo","year":"2022","journal-title":"Standard operating procedures for whole body perfsuion fixation of mice."},{"key":"B5","doi-asserted-by":"crossref","first-page":"1255","DOI":"10.1126\/science.aam9080","article-title":"Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson\u2019s disease.","volume":"357","author":"Burbulla","year":"2017","journal-title":"Science"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1186\/1741-7007-12-34","article-title":"Mitochondria as signaling organelles.","volume":"12","author":"Chandel","year":"2014","journal-title":"BMC Biol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1579","DOI":"10.1002\/jnr.24511","article-title":"Differential mitochondrial morphology in ventral striatal projection neuron subtypes.","volume":"97","author":"Chandra","year":"2019","journal-title":"J. Neurosci. Res."},{"key":"B8","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1016\/j.neuroscience.2006.01.034","article-title":"Differences in mitochondrial movement and morphology in young and mature primary cortical neurons in culture.","volume":"141","author":"Chang","year":"2006","journal-title":"Neuroscience"},{"key":"B9","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1002\/jnr.24374","article-title":"Neurons rely on glucose rather than astrocytic lactate during stimulation.","volume":"97","author":"Diaz-Garcia","year":"2019","journal-title":"J. Neurosci. Res."},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2021.109509","article-title":"3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus.","volume":"36","author":"Faitg","year":"2021","journal-title":"Cell Rep."},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.11583","article-title":"Dendritic mitochondria reach stable positions during circuit development.","volume":"5","author":"Faits","year":"2016","journal-title":"Elife"},{"key":"B12","doi-asserted-by":"publisher","first-page":"462","DOI":"10.1126\/science.3260686","article-title":"Nonoxidative glucose consumption during focal physiologic neural activity.","volume":"241","author":"Fox","year":"1988","journal-title":"Science"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.3791\/3564","article-title":"Whole animal perfusion fixation for rodents.","volume":"30","author":"Gage","year":"2012","journal-title":"J. Vis. Exp."},{"key":"B14","doi-asserted-by":"crossref","first-page":"650","DOI":"10.1038\/s41586-021-04059-0","article-title":"Disruption of mitochondrial complex I induces progressive parkinsonism.","volume":"599","author":"Gonzalez-Rodriguez","year":"2021","journal-title":"Nature"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1101\/2021.06.27.450055","article-title":"A comprehensive approach to artifact-free sample preparation and the assessment of mitochondrial morphology in tissue and cultured cells.","author":"Hinton","year":"2022","journal-title":"bioRxiv"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1186\/s13229-020-00323-8","article-title":"Absence of parvalbumin increases mitochondria volume and branching of dendrites in inhibitory PVALB neurons in vivo: A point of convergence of autism spectrum disorder (ASD) risk gene phenotypes.","volume":"11","author":"Janickova","year":"2020","journal-title":"Mol. Autism"},{"key":"B17","doi-asserted-by":"publisher","first-page":"6938","DOI":"10.1523\/JNEUROSCI.5095-13.2014","article-title":"Evidence that dendritic mitochondria negatively regulate dendritic branching in pyramidal neurons in the neocortex.","volume":"34","author":"Kimura","year":"2014","journal-title":"J. Neurosci."},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1002516","article-title":"LKB1 regulates mitochondria-dependent presynaptic calcium clearance and neurotransmitter release properties at excitatory synapses along cortical axons.","volume":"14","author":"Kwon","year":"2016","journal-title":"PLoS Biol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1016\/j.cophys.2018.03.009","article-title":"Emerging roles of mitochondria in synaptic transmission and neurodegeneration.","volume":"3","author":"Lee","year":"2018","journal-title":"Curr. Opin. Physiol."},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32130-5","article-title":"Abeta42 oligomers trigger synaptic loss through CAMKK2-AMPK-dependent effectors coordinating mitochondrial fission and mitophagy.","volume":"13","author":"Lee","year":"2022","journal-title":"Nat. Commun."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-07416-2","article-title":"MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size.","volume":"9","author":"Lewis","year":"2018","journal-title":"Nat. Commun."},{"key":"B22","doi-asserted-by":"publisher","first-page":"2602","DOI":"10.1016\/j.cub.2016.07.064","article-title":"Progressive decrease of mitochondrial motility during maturation of cortical axons in vitro and in vivo.","volume":"26","author":"Lewis","year":"2016","journal-title":"Curr. Biol."},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-13142-0","article-title":"Brain activity regulates loose coupling between mitochondrial and cytosolic Ca(2+) transients.","volume":"10","author":"Lin","year":"2019","journal-title":"Nat. Commun."},{"key":"B24","doi-asserted-by":"publisher","first-page":"314","DOI":"10.1016\/j.cell.2016.05.039","article-title":"Parkinson\u2019s disease-related proteins PINK1 and Parkin repress mitochondrial antigen presentation.","volume":"166","author":"Matheoud","year":"2016","journal-title":"Cell"},{"key":"B25","author":"Montana","year":"2022","journal-title":"Rodent brain perfusion."},{"key":"B26","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1007\/s10863-014-9575-7","article-title":"Mitochondrial dynamics: Cell-type and hippocampal region specific changes following global cerebral ischemia.","volume":"47","author":"Owens","year":"2015","journal-title":"J. Bioenerg. Biomembr."},{"key":"B27","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1002\/cne.20682","article-title":"Mitochondria form a filamentous reticular network in hippocampal dendrites but are present as discrete bodies in axons: A three-dimensional ultrastructural study.","volume":"492","author":"Popov","year":"2005","journal-title":"J. Comp. Neurol."},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.3390\/biom11050711","article-title":"The combination of paraformaldehyde and glutaraldehyde is a potential fixative for mitochondria.","volume":"11","author":"Qin","year":"2021","journal-title":"Biomolecules"},{"key":"B29","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.cell.2018.12.013","article-title":"Spatially stable mitochondrial compartments fuel local translation during plasticity.","volume":"176","author":"Rangaraju","year":"2019","journal-title":"Cell"},{"key":"B30","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1152\/physrev.1997.77.3.731","article-title":"Cellular energy utilization and molecular origin of standard metabolic rate in mammals.","volume":"77","author":"Rolfe","year":"1997","journal-title":"Physiol. Rev."},{"key":"B31","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1016\/j.bbadis.2013.09.010","article-title":"The Alzheimer\u2019s disease mitochondrial cascade hypothesis: Progress and perspectives.","volume":"1842","author":"Swerdlow","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"B32","doi-asserted-by":"publisher","first-page":"1082","DOI":"10.1016\/j.cell.2022.01.023","article-title":"Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.","volume":"185","author":"Turner","year":"2022","journal-title":"Cell"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0134205","article-title":"Anesthesia and monitoring in small laboratory mammals used in anesthesiology, respiratory and critical care research: A systematic review on the current reporting in top-10 impact factor ranked journals.","volume":"10","author":"Uhlig","year":"2015","journal-title":"PLoS One"},{"key":"B34","doi-asserted-by":"crossref","first-page":"9090","DOI":"10.1523\/JNEUROSCI.1357-09.2009","article-title":"Impaired balance of mitochondrial fission and fusion in Alzheimer\u2019s disease.","volume":"29","author":"Wang","year":"2009","journal-title":"J. Neurosci."},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.21769\/BioProtoc.3988","article-title":"Transcardiac perfusion of the mouse for brain tissue dissection and fixation.","volume":"11","author":"Wu","year":"2021","journal-title":"Bio Protoc."},{"key":"B36","doi-asserted-by":"publisher","first-page":"1368","DOI":"10.15252\/embj.201593102","article-title":"FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions.","volume":"35","author":"Wu","year":"2016","journal-title":"EMBO J."},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1038\/srep18725","article-title":"Altered brain energetics induces mitochondrial fission arrest in Alzheimer\u2019s disease.","volume":"6","author":"Zhang","year":"2016","journal-title":"Sci. Rep."},{"key":"B38","doi-asserted-by":"publisher","first-page":"1656","DOI":"10.1038\/s41590-019-0511-1","article-title":"Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance.","volume":"20","author":"Zheng","year":"2019","journal-title":"Nat. Immunol."},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.3389\/fnagi.2021.721428","article-title":"Mitochondrial dynamics: A potential therapeutic target for ischemic stroke.","volume":"13","author":"Zhou","year":"2021","journal-title":"Front. Aging Neurosci."}],"container-title":["Frontiers in Molecular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2022.1042616\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T05:15:50Z","timestamp":1667366150000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2022.1042616\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,2]]},"references-count":39,"alternative-id":["10.3389\/fnmol.2022.1042616"],"URL":"https:\/\/doi.org\/10.3389\/fnmol.2022.1042616","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2022.09.08.507147","asserted-by":"object"}]},"ISSN":["1662-5099"],"issn-type":[{"value":"1662-5099","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,2]]},"article-number":"1042616"}}