{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,16]],"date-time":"2025-09-16T18:06:03Z","timestamp":1758045963972,"version":"3.44.0"},"reference-count":39,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2025,4,25]],"date-time":"2025-04-25T00:00:00Z","timestamp":1745539200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Anim Models and Exp Med"],"published-print":{"date-parts":[[2025,7]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>Rabies virus (RABV)\u2013derived neuronal tracing tools are extensively applied in retrograde tracing due to their strict retrograde transsynaptic transfer property and low neurotoxicity. However, the RABV infection and expression of fluorescence products would be gradually cleared while the infected neurons still survive, a phenomenon known as non\u2010cytolytic immune clearance (NCLIC). This phenomenon introduced the risk of fluorescence loss and led to the omission of a subset of neurons that should be labeled, thereby interfering in the analysis of tracing results.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>To compensate for the fluorescence loss problem, in this study, we developed a novel marker footprints (MF) mouse, involving a Cre recombinase\u2010dependent red fluorescent reporter system and systemic expression of glycoprotein (G) and ASLV\u2010A receptor (TVA). Using this mouse model combined with the well\u2010developed RABV\u2010EnvA\u2010\u0394G\u2010GFP\u2010Cre viral tool, we developed a novel green\u2010to\u2010red spectral labeling strategy.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Neurons in the MF mouse could be co\u2010labeled with green fluorescence from the very quick expression of the viral tool and with red fluorescence from the relatively slow expression of the neuron itself, so neurons undergoing NCLIC with green fluorescence loss could be relabeled red. Furthermore, newly infected neurons could be labeled green and other neurons could be labeled yellow due to the temporal expression difference between the two fluorescent proteins.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>This is the first polysynaptic retrograde tracing labeling strategy that could label neurons using spectral fluorescence colors with only one injection of the viral tool, enabling its application in recognizing the labeling sequence of neurons in brain regions and enhancing the spatiotemporal resolution of neuronal tracing.<\/jats:p><\/jats:sec>","DOI":"10.1002\/ame2.70025","type":"journal-article","created":{"date-parts":[[2025,4,25]],"date-time":"2025-04-25T03:10:22Z","timestamp":1745550622000},"page":"1292-1301","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Green\u2010to\u2010red spectral labeling: A novel polysynaptic retrograde tracing strategy in the 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