Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.

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The primary general visceral nucleus of teleosts is called the commissural nucleus of Cajal (NCC). The NCC of goldfish has been divided into the medial (NCCm) and lateral (NCCl) subnuclei that receive inputs from subdiaphragmatic gastrointestinal tract and the posterior pharynx, respectively. Fiber connections of the NCC were examined by tract-tracing methods in the goldfish Carassius auratus. Tracer injections into the NCC suggested that the NCC projects directly not only to the secondary visceral sensory region in the rhombencephalic isthmus and other brain stem centers, but also to the forebrain, similar to the situations in mammals, birds, and the Nile tilapia. Although fiber connections of the NCCm and NCCl were basically similar, the NCCm was the more important source of ascending general visceral fibers to the forebrain. Topographic organization was recognized regarding projections to the isthmic secondary visceral sensory zone; input from the NCCm is represented in the secondary general visceral sensory nucleus, while input from the NCCl in the lateral edge of the secondary gustatory nucleus. Moreover, specific injections into different regions of the vagal lobe revealed that the dorsomedio-ventrolateral axis of the lobe is represented in the lateromedial axis of the secondary gustatory nucleus. These observations suggest fine topographic organization of ascending visceral sensory pathways to the isthmic secondary centers. It should also be noted that the reception of primary afferents from the posterior pharynx and projections to the secondary gustatory nucleus suggest that the NCCl may be regarded as a gustatory rather than a general visceral sensory structure.

Pseudotyped lentiviral vectors give access to pathway-selective gene manipulation via retrograde transfer. Two types of such lentiviral vectors have been developed. One is the so-called NeuRet vector pseudotyped with fusion glycoprotein type E, which preferentially transduces neurons. The other is the so-called HiRet vector pseudotyped with fusion glycoprotein type B2, which permits gene transfer into both neurons and glial cells at the injection site. Although these vectors have been applied in many studies investigating neural network functions, it remains unclear which vector is more appropriate for retrograde gene delivery in the brain. To compare the gene transfer efficiency and inflammatory response of the NeuRet vs. HiRet vectors, each vector was injected into the striatum in macaque monkeys, common marmosets, and rats. It was revealed that retrograde gene delivery of the NeuRet vector was equal to or greater than that of the HiRet vector. Furthermore, inflammation characterized by microglial and lymphocytic infiltration occurred when the HiRet vector, but not the NeuRet vector, was injected into the primate brain. The present results indicate that the NeuRet vector is more suitable than the HiRet vector for retrograde gene transfer in the primate and rodent brains.

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