Application of Virus Vectors for Anterograde Tract-Tracing and Single-Neuron Labeling Studies
Elucidating neuronal circuits is fundamental issue for understanding how the brain works and implements higher-order functions. Various viral vectors have been developed and become valuable tools for the analysis of neuronal circuits in the central nervous system. Sindbis virus vector is very useful for anterograde labeling of neurons, since the vector expresses reporter protein rapidly and strongly. Furthermore, the vector makes it possible to visualize whole structures of a single neuron by injecting adequately diluted virus solution. After immunoperoxidase staining with a tyramine-based signal amplification technique, two-dimensional reconstruction of a single neuron is performed with a virtual slide system and graphic software. In this chapter, we describe a set of single-neuron tracing method in exact detail. On the other hand, Sindbis virus vector shows very high cytotoxicity by shutting off host cellular transcription and translation and is not suitable for the experiments requiring long-term expression of transgene. In the previous study, we developed novel lentivirus vector and succeeded in neuron-specific and high-level sustained gene expression. This novel vector is expected to be applied as a sensitive anterograde tracer in addition to Sindbis virus vector.
KeywordsSindbis virus Membrane-targeting signal Anterograde tracing Single-neuron tracing Reconstruction Lentivirus Tet-Off system
This work was supported by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and Japan Society for the Promotion of Science (JSPS); Grant numbers: for Scientific Research (24500408 and 15K14333 to H.H.; 24500408 and 15H04266 to T.F.); for Exploratory Research (15K14333 to H.H.); and for Scientific Research on Innovative Areas, “Neuronal Diversity and Neocortical Organization” (25123709 to H.H.), “Foundation of Synapse and Neurocircuit Pathology” (22110007 to H.H.), and “Adaptive Circuit Shift” (15H01430 to H.H.).
- 11.Kuramoto E, Ohno S, Furuta T, Unzai T, Tanaka YR, Hioki H, Kaneko T (2015) Ventral medial nucleus neurons send thalamocortical afferents more widely and more preferentially to layer 1 than neurons of the ventral anterior-ventral lateral nuclear complex in the rat. Cereb Cortex 25:221–235CrossRefPubMedGoogle Scholar
- 14.Nakamura K, Matsumura K, Hübschle T, Nakamura Y, Hioki H, Fujiyama F, Boldogköi Z, König M, Thiel HJ, Gerstberger R, Kobayashi S, Kaneko T (2004) Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions. J Neurosci 24:5370–5380CrossRefPubMedGoogle Scholar
- 16.Ohno S, Kuramoto E, Furuta T, Hioki H, Tanaka YR, Fujiyama F, Sonomura T, Uemura M, Sugiyama K, Kaneko T (2012) A morphological analysis of thalamocortical axon fibers of rat posterior thalamic nuclei: a single neuron tracing study with viral vectors. Cereb Cortex 22:2840–2857CrossRefPubMedGoogle Scholar
- 17.Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Academic, San DiegoGoogle Scholar
- 21.Nakamura H, Hioki H, Furuta T, Kaneko T (2015) Different cortical projections from three subdivisions of the rat lateral posterior thalamic nucleus: a single-neuron tracing study with viral vectors. Eur J Neurosci. 41:1294–1310.Google Scholar