{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:45:30Z","timestamp":1785422730566,"version":"3.56.0"},"reference-count":43,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T00:00:00Z","timestamp":1775692800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007368","name":"Fondazione Pisa","doi-asserted-by":"crossref","award":["# 303\/22"],"award-info":[{"award-number":["# 303\/22"]}],"id":[{"id":"10.13039\/100007368","id-type":"DOI","asserted-by":"crossref"}]},{"award":["# 303\/22"],"award-info":[{"award-number":["# 303\/22"]}],"id":[{"id":"https:\/\/ror.org\/05jhnab13","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003196","name":"Ministero della Salute","doi-asserted-by":"publisher","award":["Ricerca Corrente 2025"],"award-info":[{"award-number":["Ricerca Corrente 2025"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]},{"award":["Ricerca Corrente 2025"],"award-info":[{"award-number":["Ricerca Corrente 2025"]}],"id":[{"id":"https:\/\/ror.org\/00789fa95","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Unbiased stereology represents the most accurate approach for estimating the total number of neurons of specific brain regions; however, its reliability critically depends on the use of rigorously defined and anatomically appropriate sampling parameters. The brain nucleus Locus Coeruleus (LC) plays a key role in several brain functions. LC impairment has been associated with a range of disorders affecting individuals across the lifespan, from infancy to adulthood. In animal models of these conditions, precise estimation of LC neuronal number is essential. The LC analysis poses specific methodological challenges due to its small size, indistinct anatomical boundaries, and age-dependent changes in neuronal density. In this study, we present a detailed and reproducible stereological workflow for the quantification of LC neurons in the mouse brain across the lifespan. Using C57BL\/6J mice at postnatal, adult, and aged stages, we optimized all key components of the Optical Fractionator method, LC neurons were identified by immunoperoxidase staining for tyrosine hydroxylase (TH) and quantified using systematic-random sampling implemented in Stereo Investigator\u00ae software. We show that age-specific adjustment of stereological parameters is necessary to obtain reliable estimates, particularly at early postnatal stages characterized by high neuronal packing density. With the optimized protocols described here, TH+ LC neuron counts consistently met accepted precision criteria, as assessed by the Gundersen coefficient of error.<\/jats:p>","DOI":"10.3390\/mps9020064","type":"journal-article","created":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T12:21:32Z","timestamp":1775737292000},"page":"64","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Stereological Assessment of Locus Coeruleus in the Mouse: A Methodological Study in Pups and Adult Animals"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-0630-1891","authenticated-orcid":false,"given":"Marco","family":"Scotto","sequence":"first","affiliation":[{"name":"Sant\u2019Anna School of Advanced Studies, 56125 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1894-4128","authenticated-orcid":false,"given":"Alessandro","family":"Galgani","sequence":"additional","affiliation":[{"name":"Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, 56125 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1211-1552","authenticated-orcid":false,"given":"Marina","family":"Boido","sequence":"additional","affiliation":[{"name":"Department of Neuroscience Rita Levi Montalcini, University of Turin, 10126 Turin, Italy"},{"name":"Neuroscience Institute Cavalieri Ottolenghi, University of Turin, 10043 Orbassano (TO), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nooria","family":"Mohammady","sequence":"additional","affiliation":[{"name":"Department of Biology, University of Pisa, 56125 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5909-2128","authenticated-orcid":false,"given":"Alessandro","family":"Vercelli","sequence":"additional","affiliation":[{"name":"Department of Neuroscience Rita Levi Montalcini, University of Turin, 10126 Turin, Italy"},{"name":"Neuroscience Institute Cavalieri Ottolenghi, University of Turin, 10043 Orbassano (TO), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2940-8128","authenticated-orcid":false,"given":"Filippo S.","family":"Giorgi","sequence":"additional","affiliation":[{"name":"Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, 56125 Pisa, Italy"},{"name":"I.R.C.C.S. Fondazione Stella Maris, Calambrone, 56128 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/s41583-020-0360-9","article-title":"Locus Coeruleus: A New Look at the Blue Spot","volume":"21","author":"Poe","year":"2020","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1007\/s00702-021-02338-8","article-title":"The Connections of Locus Coeruleus with Hypothalamus: Potential Involvement in Alzheimer\u2019s Disease","volume":"128","author":"Giorgi","year":"2021","journal-title":"J. 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