Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Takeshi Imai Last modified date:2021.10.22

Professor / Department of Basic Medicine / Faculty of Medical Sciences


Papers
1. A Tsuboi, T Miyazaki, T Imai, H Sakano, Olfactory sensory neurons expressing class I odorant receptors converge their axons on an antero-dorsal domain of the olfactory bulb in the mouse, EUROPEAN JOURNAL OF NEUROSCIENCE, 10.1111/j.1460-9568.2006.04675.x, 23, 6, 1436-1444, 2006.03, Vertebrate odorant receptor (OR) genes are divided phylogenetically into two distinct classes: the fish-like class I and the terrestrial-specific class II. In the present study, we systematically analysed mouse class I OR genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (OE) and the projection sites of their olfactory sensory neurons (OSNs) in the olfactory bulb (OB). In situ hybridization (ISH) revealed that most class I OR genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the OE. Furthermore, there appeared to be no significant differences in the distributions of OSNs expressing class I genes within zone 1. These results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the OE. Some class I ORs are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. Our ISH analysis has revealed that OSNs expressing the class I ORs in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the OB..
2. Takeshi Imai, Misao Suzuki, Hitoshi Sakano, Odorant receptor-derived cAMP signals direct axonal targeting, SCIENCE, 10.1126/science.1131794, 314, 5799, 657-661, 2006.10, In mammals, odorant receptors (ORs) direct the axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. We show that cyclic adenosine monophosphate ( cAMP) signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, stimulatory G protein, cAMP-dependent protein kinase, and cAMP response element - binding protein shifted the axonal projection sites along the anterior-posterior axis in the olfactory bulb. Thus, it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determine the target destinations of OSNs..
3. Takeshi Imai, Hitoshi Sakano, Roles of odorant receptors in projecting axons in the mouse olfactory system, CURRENT OPINION IN NEUROBIOLOGY, 10.1016/j.conb.2007.08.002, 17, 5, 507-515, 2007.10, In the mouse olfactory epithelium, there are about ten million olfactory sensory neurons, each expressing a single type of odorant receptor out of similar to 1000. Olfactory sensory neurons expressing the same odorant receptor converge their axons to a specific set of glomeruli on the olfactory bulb. How odorant receptors play an instructive role in the projection of axons to the olfactory bulb has been one of the major issues of developmental neurobiology. Recent studies revealed previously overlooked roles of odorant receptor-derived cAMP signals in the axonal projection of olfactory sensory neurons; the levels of cAMP and neuronal activity appear to determine the expression levels of axon guidance/sorting molecules and thereby direct the axonal projection of olfactory sensory neurons. These findings provide new insights as to how peripheral inputs instruct neuronal circuit formation in the mammalian brain..
4. Takeshi Imai, Hitoshi Sakano, Odorant receptor-mediated signaling in the mouse, CURRENT OPINION IN NEUROBIOLOGY, 10.1016/j.conb.2008.07.009, 18, 3, 251-260, 2008.06, In the mouse olfactory system, there are similar to 1000 types of odorant receptors (ORs), which perform multiple functions in olfactory sensory neurons (OSNs). In addition to detecting odors, the functional OR protein ensures the singular gene choice of the OR by negative-feedback regulation. ORs also direct the axonal projection of OSNs both globally and locally by modulating the transcriptional levels of axon-guidance and axon-sorting molecules. In these latter processes, the second messenger, cAMP. plays differential roles in the fasciculation and targeting of axons. In this review, we will discuss how ORs differentially regulate intracellular signals for distinct functions..
5. Takeshi Imai, Takahiro Yamazaki, Reiko Kobayakawa, Ko Kobayakawa, Takaya Abe, Misao Suzuki, Hitoshi Sakano, Pre-Target Axon Sorting Establishes the Neural Map Topography, SCIENCE, 10.1126/science.1173596, 325, 5940, 585-590, 2009.07, Sensory information detected by the peripheral nervous system is represented as a topographic map in the brain. It has long been thought that the topography of the map is determined by graded positional cues that are expressed by the target. Here, we analyzed the pre-target axon sorting for olfactory map formation in mice. In olfactory sensory neurons, an axon guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, are expressed in a complementary manner. We found that expression levels of Neuropilin-1 determined both pre-target sorting and projection sites of axons. Olfactory sensory neuron-specific knockout of Semaphorin-3A perturbed axon sorting and altered the olfactory map topography. Thus, pre-target axon sorting plays an important role in establishing the topographic order based on the relative levels of guidance molecules expressed by axons..
6. Takeshi Imai, Hitoshi Sakano, Leslie B. Vosshall, Topographic Mapping-The Olfactory System, COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY, 10.1101/cshperspect.a001776, 2, 8, a001776, 2010.08, Sensory systems must map accurate representations of the external world in the brain. Although the physical senses of touch and vision build topographic representations of the spatial coordinates of the body and the field of view, the chemical sense of olfaction maps discontinuous features of chemical space, comprising an extremely large number of possible odor stimuli. In both mammals and insects, olfactory circuits are wired according to the convergence of axons from sensory neurons expressing the same odorant receptor. Synapses are organized into distinctive spherical neuropils-the olfactory glomeruli-that connect sensory input with output neurons and local modulatory interneurons. Although there is a strong conservation of form in the olfactory maps of mammals and insects, they arise using divergent mechanisms. Olfactory glomeruli provide a unique solution to the problem of mapping discontinuous chemical space onto the brain..
7. Akio Tsuboi, Takeshi Imai, Hiroyuki K. Kato, Hideyuki Matsumoto, Kei M. Igarashi, Misao Suzuki, Kensaku Mori, Hitoshi Sakano, Two highly homologous mouse odorant receptors encoded by tandemly-linked MOR29A and MOR29B genes respond differently to phenyl ethers, EUROPEAN JOURNAL OF NEUROSCIENCE, 10.1111/j.1460-9568.2010.07495.x, 33, 2, 205-213, 2011.01, Since the discovery of odorant receptors (ORs) in rodents, most ORs have remained orphan receptors. Even for deorphanized ORs in vitro, their in vivo properties are largely unknown. Here, we report odor response profiles of two highly homologous mouse ORs, MOR29A and MOR29B, both in vivo and in vitro. The BAC transgenic mouse was generated, in which olfactory sensory neurons (OSNs) expressing the transgenes MOR29A and MOR29B were differently tagged with IRES-gapECFP and IRES-gapEYFP, respectively. MOR29A- and MOR29B-expressing OSN axons converged on separate but nearby loci on the dorsal surface of the olfactory bulb (OB). Optical imaging of intrinsic signals in the OB identified five different phenyl ethers as candidate ligands for MOR29B. Based on in vitro calcium imaging with the isolated OSNs and luciferase assay with heterologous cells, only guaiacol and vanillin were found to be potent agonists for MOR29A and MOR29B. Because of its accessible glomerular locations in the dorsal OB and defined odor response profiles both in vivo and in vitro, the MOR29A/29B tagging mouse will serve as an excellent tool for studying both odor-signal processing and neural circuitry in the OB..
8. Takeshi Imai, Hitoshi Sakano, Axon-axon interactions in neuronal circuit assembly: lessons from olfactory map formation, EUROPEAN JOURNAL OF NEUROSCIENCE, 10.1111/j.1460-9568.2011.07817.x, 34, 10, 1647-1654, 2011.11, During the development of the nervous system, neurons often connect axons and dendrites over long distances, which are navigated by chemical cues. During the past few decades, studies on axon guidance have focused on chemical cues provided by the axonal target or intermediate target. However, recent studies have shed light on the roles and mechanisms underlying axonaxon interactions during neuronal circuit assembly. The roles of axonaxon interactions are best exemplified in recent studies on olfactory map formation in vertebrates. Pioneerfollower interaction is essential for the axonal pathfinding process. Pre-target axon sorting establishes the anteriorposterior map order. The temporal order of axonal projection is converted to dorsalventral topography with the aid of secreted molecules provided by early-arriving axons. An activity-dependent process to form a discrete map also depends on axon sorting. Thus, an emerging principle of olfactory map formation is the self-organisation of axons rather than the lock and key matching between axons and targets. In this review, we discuss how axonaxon interactions contribute to neuronal circuit assembly..
9. Takeshi Imai, Positional information in neural map development: Lessons from the olfactory system, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/j.1440-169X.2012.01334.x, 54, 3, 358-365, 2012.04, Positional information is fundamental in development. Although molecular gradients are thought to represent positional information in various systems, the molecular logic used to interpret these gradients remains controversial. In the nervous system, sensory maps are formed in the brain based on gradients of axon guidance molecules. However, it remains unclear how axons find their targets based on relative, not absolute, expression levels of axon guidance receptors. No model solely based on axontarget interactions explains this point. Recent studies in the olfactory system suggested that the neural map formation requires axonaxon interactions, which is known as axon sorting. This review discusses how axonaxon and axontarget interactions interpret molecular gradients and determine the axonal projection sites in neural map formation..
10. Meng-Tsen Ke, Satoshi Fujimoto, Takeshi Imai, SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction, NATURE NEUROSCIENCE, 10.1038/nn.3447, 16, 8, 1154-U246, 2013.08, We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains..
11. Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity..
12. Yohei Sasagawa, Itoshi Nikaido, Tetsutaro Hayashi, Hiroki Danno, Kenichiro D. Uno, Takeshi Imai, Hiroki R Ueda, Quartz-Seq: A highly reproducible and sensitive single-cell RNA sequencing method, reveals nongenetic gene-expression heterogeneity, Genome Biology, 10.1186/gb-2013-14-4-r31, 14, 4, R31, 2013.04, Development of a highly reproducible and sensitive single-cell RNA sequencing (RNA-seq) method would facilitate the understanding of the biological roles and underlying mechanisms of non-genetic cellular heterogeneity. In this study, we report a novel single-cell RNA-seq method called Quartz-Seq that has a simpler protocol and higher reproducibility and sensitivity than existing methods. We show that single-cell Quartz-Seq can quantitatively detect various kinds of non-genetic cellular heterogeneity, and can detect different cell types and different cell-cycle phases of a single cell type. Moreover, this method can comprehensively reveal gene-expression heterogeneity between single cells of the same cell type in the same cell-cycle phase..
13. Ai Nakashima, Haruki Takeuchi, Takeshi Imai, Harumi Saito, Hiroshi Kiyonari, Takaya Abe, Min Chen, Lee S. Weinstein, C. Ron Yu, Daniel R. Storm, Hirofumi Nishizumi, Hitoshi Sakano, Agonist-Independent GPCR Activity Regulates Anterior-Posterior Targeting of Olfactory Sensory Neurons, CELL, 10.1016/j.cell.2013.08.033, 154, 6, 1314-1325, 2013.09, G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the beta 2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules-e.g., Neuropilin-1 and Plexin-A1-but not of glomerular segregation molecules-e.g., Kirrel2 and Kirrel3-thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that G(s), but not G(olf), is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules..
14. Hiroyuki Takaba, Takeshi Imai, Shoji Miki, Yasuyuki Morishita, Akihiro Miyashita, Naoko Ishikawa, Hirofumi Nishizumi, Hitoshi Sakano, A major allogenic leukocyte antigen in the agnathan hagfish, Scientific Reports, 10.1038/srep01716, 3, 1716, 2013.04, All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity..
15. Meng-Tsen Ke, Satoshi Fujimoto, Takeshi Imai, Optical Clearing Using SeeDB., Bio-protocol, 4, 3, e1042, 2014.02.
16. Takeshi Imai, Construction of functional neuronal circuitry in the olfactory bulb, SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY, 10.1016/j.semcdb.2014.07.012, 35, 180-188, 2014.11, Recent studies using molecular genetics, electrophysiology, in vivo imaging, and behavioral analyses have elucidated detailed connectivity and function of the mammalian olfactory circuits. The olfactory bulb is the first relay station of olfactory perception in the brain, but it is more than a simple relay: olfactory information is dynamically tuned by local olfactory bulb circuits and converted to spatiotemporal neural code for higher-order information processing. Because the olfactory bulb processes 1000 discrete input channels from different odorant receptors, it serves as a good model to study neuronal wiring specificity, from both functional and developmental aspects. This review summarizes our current understanding of the olfactory bulb circuitry from functional standpoint and discusses important future studies with particular focus on its development and plasticity. (C) 2014 The Authors. Published by Elsevier Ltd..
17. Meng-Tsen Ke, Yasuhiro Nakai, Satoshi Fujimoto, Rie Takayama, Shuhei Yoshida, Tomoya S. Kitajima, Makoto Sato, Takeshi Imai, Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent, CELL REPORTS, 10.1016/j.celrep.2016.02.057, 14, 11, 2718-2732, 2016.03, Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of > 100 mu m, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels..
18. Aya Murai, Ryo Iwata, Satoshi Fujimoto, Shuhei Aihara, Akio Tsuboi, Yuko Muroyama, Tetsuichiro Saito, Kazunori Nishizaki, Takeshi Imai, Distorted coarse axon targeting and reduced dendrite connectivity underlie Dysosmia after olfactory axon injury, eNeuro, 10.1523/ENEURO.0242-16.2016, 3, 5, 2016.09, The glomerular map in the olfactory bulb (OB) is the basis for odor recognition. Once established during development, the glomerular map is stably maintained throughout the life of an animal despite the continuous turnover of olfactory sensory neurons (OSNs). However, traumatic damage to OSN axons in the adult often leads to dysosmia, a qualitative and quantitative change in olfaction in humans. A mouse model of dysosmia has previously indicated that there is an altered glomerular map in the OB after the OSN axon injury
however, the underlying mechanisms that cause the map distortion remain unknown. In this study, we examined how the glomerular map is disturbed and how the odor information processing in the OB is affected in the dysosmia model mice. We found that the anterior-posterior coarse targeting of OSN axons is disrupted after OSN axon injury, while the local axon sorting mechanisms remained. We also found that the connectivity of mitral/tufted cell dendrites is reduced after injury, leading to attenuated odor responses in mitral/tufted cells. These results suggest that existing OSN axons are an essential scaffold for maintaining the integrity of the olfactory circuit, both OSN axons and mitral/tufted cell dendrites, in the adult..
19. Yohei Sasagawa, Itoshi Nikaido, Tetsutaro Hayashi, Hiroki Danno, Kenichiro D Uno, Takeshi Imai, Hiroki R. Ueda, Erratum to: Quartz-Seq: A highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity. [Genome Biol. 14, (2013), (R31)], Genome Biology, 10.1186/s13059-017-1154-x, 18, 1, 9, 2017.01, After publication of our article [1], we noticed some errors. In this manuscript, we amplified cDNA with 41.67 nmol/l RT primer and 70 nmol/l tagging primer for single-cell Quartz-Seq, and not pmol/l as originally stated. In addition, two mathematical expressions were inadvertently omitted. Thus, in the section Whole-transcript amplification for single-cell Quartz-Seq, the following are correct: Immediately after the second centrifugation, 0.8 μl of priming buffer (1.5× PCR buffer with MgCl2 (TaKaRa Bio), 41.67 nmol/l of the RT primer (HPLC-purified
Table 1), 4 U/μl of RNase inhibitor (RNasin Plus
Promega Corp., Madison, WI, USA), and 50 μmol/l dNTPs were added to each tube. And We then added 23 μl of the second-strand buffer (1.09× MightyAmp Buffer v2 (TaKaRa), 70 nmol/l tagging primer (HPLC-purified
Table 1), and 0.054 U/μl MightyAmp DNA polymerase (TaKaRa)) to each tube. In the section Bioinformatics analysis, the equations should appear as follows: The MI is considered the Kullback-Leibler distance from the joint probability density to the product of the marginal probability densities as follows: The MI is always non-negative, symmetric, and equal to 0 only if × and Y are independent. The MI can be represented as a summation of entropies..
20. Meng-Tsen Ke, Takeshi Imai, Optical Clearing and Index Matching of Tissue Samples for High-resolution Fluorescence Imaging Using SeeDB2, Bio-protocol, 8, 20, e3046, 2018.10.
21. In vivo Optical Access to Olfactory Sensory Neurons in the Mouse Olfactory Epithelium.
22. Development of the olfactory system: from sensory neurons to cortical projections.
23. Aihara S, Fujimoto S, Sakaguchi R, Imai T., BMPR-2 gates activity-dependent stabilization of primary dendrites during mitral cell remodeling, Cell Reports, https://doi.org/10.1016/j.celrep.2021.109276, 12, 22, 109276, 2021.06, Developing neurons initially form excessive neurites and then remodel them based on molecular cues and neuronal activity. Developing mitral cells in the olfactory bulb initially extend multiple primary dendrites. They then stabilize single primary dendrites while eliminating others. However, the mechanisms underlying selective dendrite remodeling remain elusive. Using CRISPR-Cas9-based knockout screening combined with in utero electroporation, we identify BMPR-2 as a key regulator for selective dendrite stabilization. Bmpr2 knockout and its rescue experiments show that BMPR-2 inhibits LIMK without ligands and thereby permits dendrite destabilization. In contrast, the overexpression of antagonists and agonists indicates that ligand-bound BMPR-2 stabilizes dendrites, most likely by releasing LIMK. Using genetic and FRET imaging experiments, we demonstrate that free LIMK is activated by NMDARs via Rac1, facilitating dendrite stabilization through F-actin formation. Thus, the selective stabilization of primary dendrites is ensured by concomitant inputs of BMP ligands and neuronal activity..
24. Inagaki S, Iwata R, Iwamoto M, Imai T., Widespread inhibition, antagonism, and synergy in mouse olfactory sensory neurons in vivo, Cell Reports, https://doi.org/10.1016/j.celrep.2020.107814, 13, 107814, 107814, 2020.06, Sensory information is selectively or non-selectively enhanced and inhibited in the brain, but it remains unclear whether and how this occurs at the most peripheral level. Using in vivo calcium imaging of mouse olfactory bulb and olfactory epithelium in wild-type and mutant animals, we show that odors produce not only excitatory but also inhibitory responses in olfactory sensory neurons (OSNs). Heterologous assays indicate that odorants can act as agonists to some but inverse agonists to other odorant receptors. We also demonstrate that responses to odor mixtures are extensively suppressed or enhanced in OSNs. When high concentrations of odors are mixed, widespread antagonism suppresses the overall response amplitudes and density. In contrast, a mixture of low concentrations of odors often produces synergistic effects and boosts the faint odor inputs. Thus, odor responses are extensively tuned by inhibition, antagonism, and synergy at the most peripheral level, contributing to robust sensory representations..
25. Ryo Iwata, Hiroshi Kiyonari, Takeshi Imai, Mechanosensory-Based Phase Coding of Odor Identity in the Olfactory Bulb, Neuron, 10.1016/j.neuron.2017.11.008, 96, 5, 1139-1152.e7, 2017.12, Mitral and tufted (M/T) cells in the olfactory bulb produce rich temporal patterns of activity in response to different odors. However, it remains unknown how these temporal patterns are generated and how they are utilized in olfaction. Here we show that temporal patterning effectively discriminates between the two sensory modalities detected by olfactory sensory neurons (OSNs): odor and airflow-driven mechanical signals. Sniff-induced mechanosensation generates glomerulus-specific oscillatory activity in M/T cells, whose phase was invariant across airflow speed. In contrast, odor stimulation caused phase shifts (phase coding). We also found that odor-evoked phase shifts are concentration invariant and stable across multiple sniff cycles, contrary to the labile nature of rate coding. The loss of oscillatory mechanosensation impaired the precision and stability of phase coding, demonstrating its role in olfaction. We propose that phase, not rate, coding is a robust encoding strategy of odor identity and is ensured by airflow-induced mechanosensation in OSNs. Iwata et al. demonstrate that phase coding, but not rate coding, in mitral cells is useful for concentration-invariant odor identity coding. They also found that mechanosensation in olfactory sensory neurons facilitates, rather than masks, the robust phase coding of odors..
26. Richi Sakaguchi, Marcus N. Leiwe, Takeshi Imai, Bright multicolor labeling of neuronal circuits with fluorescent proteins and chemical tags, ELIFE, 10.7554/eLife.40350, 7, 2018.11, The stochastic multicolor labeling method 'Brainbow' is a powerful strategy to label multiple neurons differentially with fluorescent proteins; however, the fluorescence levels provided by the original attempts to use this strategy were inadequate. In the present study, we developed a stochastic multicolor labeling method with enhanced expression levels that uses a tetracycline-operator system (Tetbow). We optimized Tetbow for either plasmid or virus vector-mediated multicolor labeling. When combined with tissue clearing, Tetbow was powerful enough to visualize the three-dimensional architecture of individual neurons. Using Tetbow, we were able to visualize the axonal projection patterns of individual mitral/tufted cells along several millimeters in the mouse olfactory system. We also developed a Tetbow system with chemical tags, in which genetically encoded chemical tags were labeled with synthetic fluorophores. This was useful in expanding the repertoire of the fluorescence labels and the applications of the Tetbow system. Together, these new tools facilitate light-microscopy-based neuronal tracing at both a large scale and a high resolution..