{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T05:49:13Z","timestamp":1787636953191,"version":"build-2736575974"},"reference-count":45,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2021,8,25]],"date-time":"2021-08-25T00:00:00Z","timestamp":1629849600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["15K15202"],"award-info":[{"award-number":["15K15202"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["15H05724"],"award-info":[{"award-number":["15H05724"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["15KK025708"],"award-info":[{"award-number":["15KK025708"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["16H06280"],"award-info":[{"award-number":["16H06280"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21H02520"],"award-info":[{"award-number":["21H02520"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["20K15837"],"award-info":[{"award-number":["20K15837"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21H00428"],"award-info":[{"award-number":["21H00428"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"The Suzuken Memorial Foundation","award":["19-085"],"award-info":[{"award-number":["19-085"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at \u221225 \u00b0C for several years (4\u20136 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.<\/jats:p>","DOI":"10.3390\/ijms22179180","type":"journal-article","created":{"date-parts":[[2021,8,26]],"date-time":"2021-08-26T00:19:26Z","timestamp":1629937166000},"page":"9180","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at \u221225 \u00b0C for Several Years"],"prefix":"10.3390","volume":"22","author":[{"given":"Akito","family":"Otubo","sequence":"first","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sho","family":"Maejima","sequence":"additional","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7216-443X","authenticated-orcid":false,"given":"Takumi","family":"Oti","sequence":"additional","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"},{"name":"Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6129-7029","authenticated-orcid":false,"given":"Keita","family":"Satoh","sequence":"additional","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"},{"name":"Department of Anatomy, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasumasa","family":"Ueda","sequence":"additional","affiliation":[{"name":"Department of Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan"},{"name":"Department of Physiology, Kansai Medical University, Shinmachi, Hirakata, Osaka 573-1191, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John F.","family":"Morris","sequence":"additional","affiliation":[{"name":"Department of Physiology, Anatomy & Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatsuya","family":"Sakamoto","sequence":"additional","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6224-4591","authenticated-orcid":false,"given":"Hirotaka","family":"Sakamoto","sequence":"additional","affiliation":[{"name":"Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan"},{"name":"Department of Physiology, Anatomy & Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1038\/506024a","article-title":"Biomedicine: The changing face of primate research","volume":"506","author":"Abbott","year":"2014","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1038\/s41583-018-0072-6","article-title":"The neural mechanisms and circuitry of the pair bond","volume":"19","author":"Walum","year":"2018","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/S0079-6123(08)64379-4","article-title":"Vasopressin and oxytocin: Distribution and putative functions in the brain","volume":"60","author":"Buijs","year":"1983","journal-title":"Prog. Brain Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/0167-0115(93)90175-8","article-title":"Structure and folding properties of neurophysin and its peptide complexes: Biological implications","volume":"45","author":"Breslow","year":"1993","journal-title":"Regul. Pept."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1046\/j.1365-2826.2003.00865.x","article-title":"Further delineation of the sequences required for the expression and physiological regulation of the vasopressin gene in transgenic rat hypothalamic magnocellular neurones","volume":"15","author":"Davies","year":"2003","journal-title":"J. Neuroendocrinol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1038\/295299a0","article-title":"Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor","volume":"295","author":"Land","year":"1982","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.1152\/physrev.00035.2011","article-title":"Vasopressin V1a and V1b receptors: From molecules to physiological systems","volume":"92","author":"Koshimizu","year":"2012","journal-title":"Physiol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/978-3-030-22989-4_5","article-title":"Neurosecretory Vesicles: Structure, Distribution, Release and Breakdown","volume":"Volume 8","author":"Lemons","year":"2020","journal-title":"Neurosecretion: Secretory Mechanisms"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1394","DOI":"10.1126\/science.6267699","article-title":"Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin","volume":"213","author":"Vale","year":"1981","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/0196-9781(84)90261-4","article-title":"The CRF neuron: Immunocytochemical study","volume":"5","author":"Fellmann","year":"1984","journal-title":"Peptides"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4054","DOI":"10.1210\/en.2014-1182","article-title":"Visualization of corticotropin-releasing factor neurons by fluorescent proteins in the mouse brain and characterization of labeled neurons in the paraventricular nucleus of the hypothalamus","volume":"155","author":"Itoi","year":"2014","journal-title":"Endocrinology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1159\/000126339","article-title":"Colocalization of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of the human hypothalamus","volume":"57","author":"Mouri","year":"1993","journal-title":"Neuroendocrinology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/317248a0","article-title":"Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles","volume":"317","author":"Whitnall","year":"1985","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e12875","DOI":"10.1111\/jne.12875","article-title":"Vasopressin gene products are colocalised with corticotrophin-releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata)","volume":"32","author":"Otubo","year":"2020","journal-title":"J. Neuroendocrinol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1126\/science.182.4115.925","article-title":"Vasopressin and neurophysin: High concentrations in monkey hypophyseal portal blood","volume":"182","author":"Zimmerman","year":"1973","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/0006-8993(91)91454-9","article-title":"Ultrastructural distribution of glutamate immunoreactivity within neurosecretory endings and pituicytes of the rat neurohypophysis","volume":"564","author":"Meeker","year":"1991","journal-title":"Brain Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/0306-4522(89)90318-7","article-title":"Light and electron microscopic localization of glutamate immunoreactivity in the supraoptic nucleus of the rat hypothalamus","volume":"33","author":"Meeker","year":"1989","journal-title":"Neuroscience"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1016\/S0306-4522(01)00279-2","article-title":"Corticotropin-releasing factor is preferentially colocalized with excitatory rather than inhibitory amino acids in axon terminals in the peri-locus coeruleus region","volume":"106","author":"Valentino","year":"2001","journal-title":"Neuroscience"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1016\/S0022-5320(66)80110-7","article-title":"The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. II. Effects of varying osmolality, ionic strength, buffer system and fixative concentration of glutaraldehyde solutions","volume":"15","author":"Maunsbach","year":"1966","journal-title":"J. Ultrastruct. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1016\/S0022-5320(66)80109-0","article-title":"The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives","volume":"15","author":"Maunsbach","year":"1966","journal-title":"J. Ultrastruct. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.jchemneu.2013.02.007","article-title":"Differential expression of vesicular glutamate transporters 1 and 2 may identify distinct modes of glutamatergic transmission in the macaque visual system","volume":"50\u201351","author":"Balaram","year":"2013","journal-title":"J. Chem. Neuroanat."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1002\/cne.25026","article-title":"Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin","volume":"529","author":"Kawakami","year":"2021","journal-title":"J. Comp. Neurol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2488","DOI":"10.1111\/febs.13291","article-title":"In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: Response to osmotic stimulation","volume":"282","author":"Satoh","year":"2015","journal-title":"FEBS J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1883","DOI":"10.1073\/pnas.81.6.1883","article-title":"Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat","volume":"81","author":"Sawchenko","year":"1984","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1002\/cne.902760208","article-title":"Distribution of corticotropin-releasing-factor-like immunoreactivity in brainstem of two monkey species (Saimiri sciureus and Macaca fascicularis): An immunohistochemical study","volume":"276","author":"Foote","year":"1988","journal-title":"J. Comp. Neurol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1016\/0306-4522(95)00186-M","article-title":"Oestradiol acutely stimulates exocytosis of oxytocin and vasopressin from dendrites and somata of hypothalamic magnocellular neurons","volume":"68","author":"Wang","year":"1995","journal-title":"Neuroscience"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1007\/BF00221868","article-title":"Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques","volume":"243","author":"Castel","year":"1986","journal-title":"Cell Tissue Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1016\/0306-4522(88)90078-4","article-title":"The neurophysin-containing innervation of the forebrain of the mouse","volume":"24","author":"Castel","year":"1988","journal-title":"Neuroscience"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e11396","DOI":"10.7554\/eLife.11396","article-title":"An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence","volume":"4","author":"Frahm","year":"2015","journal-title":"eLife"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1038\/nn.4503","article-title":"Cadherins mediate cocaine-induced synaptic plasticity and behavioral conditioning","volume":"20","author":"Mills","year":"2017","journal-title":"Nat. Neurosci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1002\/cne.21478","article-title":"Metabolic and functional profiling of the normal rat retina","volume":"505","author":"Sun","year":"2007","journal-title":"J. Comp. Neurol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1002\/cne.21771","article-title":"Plasticity of lumbosacral propriospinal neurons is associated with the development of autonomic dysreflexia after thoracic spinal cord transection","volume":"509","author":"Hou","year":"2008","journal-title":"J. Comp. Neurol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/BF00305474","article-title":"Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macaca fuscata)","volume":"165","author":"Kawata","year":"1982","journal-title":"Anat. Embryol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1677\/joe.0.0570531","article-title":"Changes in the appearance of hypothalamo-neurohypophysial neurosecretory granules associated with their maturation","volume":"57","author":"Cannata","year":"1973","journal-title":"J. Endocrinol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1002\/cne.10257","article-title":"Distribution of vesicular glutamate transporter mRNA in rat hypothalamus","volume":"448","author":"Ziegler","year":"2002","journal-title":"J. Comp. Neurol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e12831","DOI":"10.1111\/jne.12831","article-title":"VGLUT-VGAT expression delineates functionally specialised populations of vasopressin-containing neurones including a glutamatergic perforant path-projecting cell group to the hippocampus in rat and mouse brain","volume":"32","author":"Zhang","year":"2020","journal-title":"J. Neuroendocrinol."},{"key":"ref_37","first-page":"182","article-title":"Glutamatergic phenotype of hypothalamic neurosecretory systems: A novel aspect of central neuroendocrine regulation","volume":"60","author":"Hrabovszky","year":"2007","journal-title":"Ideggyogy Sz."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1159\/000126843","article-title":"Glutamate: A major excitatory transmitter in neuroendocrine regulation","volume":"61","author":"Brann","year":"1995","journal-title":"Neuroendocrinology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1016\/j.neuint.2005.12.013","article-title":"Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus","volume":"48","author":"Hrabovszky","year":"2006","journal-title":"Neurochem. Int."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1515\/bchm3.1986.367.2.695","article-title":"The neurohypophyseal hormones vasopressin and oxytocin. Precursor structure, synthesis and regulation","volume":"367","author":"Rehbein","year":"1986","journal-title":"Biol. Chem. Hoppe-Seyler"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1016\/0169-328X(90)90045-F","article-title":"Structure of mouse vasopressin and oxytocin genes","volume":"8","author":"Hara","year":"1990","journal-title":"Brain Res. Mol. Brain Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"650","DOI":"10.1159\/000442015","article-title":"Age and Long-Term Hormone Treatment Effects on the Ultrastructural Morphology of the Median Eminence of Female Rhesus Macaques","volume":"103","author":"Naugle","year":"2016","journal-title":"Neuroendocrinology"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1007\/BF01045120","article-title":"Comparative Observations on Inclusions in Nerve Fibres of the Mammalian Neurohypophysis","volume":"64","author":"Holmes","year":"1964","journal-title":"Z Zellforsch Mikrosk Anat."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/0006-8993(91)91198-A","article-title":"Separate ultrastructural distributions of neurophysin and C-terminal glycopeptide within dense core vesicles in rat neural lobe","volume":"562","author":"Armstrong","year":"1991","journal-title":"Brain Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1677\/joe.0.0680209","article-title":"Hormone storage in individual neurosecretory granules of the pituitary gland: A quantitative ultrastructural approach to hormone storage in the neural lobe","volume":"68","author":"Morris","year":"1976","journal-title":"J. Endocrinol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/17\/9180\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:51:34Z","timestamp":1760165494000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/17\/9180"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,25]]},"references-count":45,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["ijms22179180"],"URL":"https:\/\/doi.org\/10.3390\/ijms22179180","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.06.07.447309","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,25]]}}}