The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.

The cortico–basal ganglia–thalamo–cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative^1–4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex⁵. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico–basal ganglia–thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico–basal ganglia–thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.

In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP⁺ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo.

Short-term memory deficits have been associated with prefrontal cortex (PFC) dysfunction in Alzheimer’s disease (AD) and AD mouse models. Extratelencephalic projection (ET) neurons in the PFC play a key role in short-term working memory, but the mechanism between ET neuronal dysfunction in the PFC and short-term memory impairment in AD is not well understood. Here, using fiber photometry and optogenetics, we found reduced neural activity in the ET neurons in the medial prefrontal cortex (mPFC) of the 5×FAD mouse model led to object recognition memory (ORM) deficits. Activation of ET neurons in the mPFC of 5×FAD mice rescued ORM impairment, and inhibition of ET neurons in the mPFC of wild type mice impaired ORM expression. ET neurons in the mPFC that project to supramammillary nucleus were necessary for ORM expression. Viral tracing and in vivo recording revealed that mPFC ET neurons received fewer cholinergic inputs from the basal forebrain in 5×FAD mice. Furthermore, activation of cholinergic fibers in the mPFC rescued ORM deficits in 5×FAD mice, while acetylcholine deficiency reduced the response of ET neurons in the mPFC to familiar objects. Taken together, our results revealed a neural mechanism behind ORM impairment in 5×FAD mice.