Significance: The cholinergic system plays a critical role in neural modulation of the mammalian brain. Here, we generated a comprehensive atlas of the cholinergic system in the mouse brain via the whole-brain imaging and reconstruction system. In the whole-brain dataset, the cholinergic neurons were divided into three categories including cortical VIP neurons, long-range projection neurons, and brainstem motor neurons. After reconstructing the cholinergic neurons in a subregion of basal forebrain, we found that their projections to the forebrain and midbrain showed neuronal subgroups with distinct projection specificity. Our work presents three-dimensional information about the cholinergic system in the mouse brain, facilitating further studies of the cholinergic system.

Resin embedding has been widely used for precise imaging of fluorescently labeled biological samples with optical and electron microscopy. The low preservation rate of fluorescence, especially for red fluorescent proteins, has limited the application of resin embedding in multifluorescent protein-labeled samples. Here, we optimized the embedding method to retain the intensity of multiple fluorescent proteins during resin embedding. By reducing the polymerization temperature from 50 to 35∘C and adding a fluorescent protein protection reagent during the embedding process, we successfully increased the fluorescence preservation rate by nearly twofold for red fluorescent proteins, including tdTomato, mCherry, and DsRed. Meanwhile, the background fluorescence decreased significantly in the optimized embedding method. This method is suitable not only for red fluorescent protein-labeled samples but also for blue (BFP) and green fluorescent protein (GFP)-labeled samples. We embedded brains labeled with BFP, DsRed, and GFP via AAV and rabies virus and acquired the distribution of input neurons to different cortical areas. With GFP/tdTomato double-labeled samples in resin, we obtained the cholinergic projectome of the pedunculopontine tegmental nucleus (PPTg) and the distribution of cholinergic neurons at single-neuron resolution in the whole brain simultaneously. Input cholinergic terminals from the PPTg were found to innervate the cholinergic soma and fiber in the neocortex, basal forebrain and brainstem, indicating that local cholinergic neurons received long-range cholinergic modulation from the midbrain. Our optimized method is useful for embedding multicolor fluorescent protein-labeled samples to acquire multidimensional structural information on neural circuits at single-neuron resolution in the whole brain.

We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

Understanding the developmental changes that affect neurons is a key step in exploring the assembly and maturation of neural circuits in the brain. For decades, researchers have used a number of labeling techniques to visualize neuronal morphology at different stages of development. However, the efficiency and accuracy of neuronal labeling technologies are limited by the complexity and fragility of neonatal brains. In this review, we illustrate the various labeling techniques utilized for examining the neurogenesis and morphological changes occurring during the early stages of development. We compare the advantages and limitations of each technique from different aspects. Then, we highlight the gaps remaining in our understanding of the structure of neurons in the neonatal mouse brain.

Neuronal senescence-like states are increasingly implicated in age-related neurodegeneration, yet the neuron-intrinsic regulators that drive these phenotypes remain poorly defined. Guided by prior transcriptomic analysis of aged basal forebrain cholinergic neurons, we investigated Srebf2 and Zmiz1 using primary basal forebrain neuronal cultures with adeno-associated virus-mediated gain- and loss-of-function, quantitative immunocytochemistry, and low-input transcriptomic profiling of fluorescence-activated cell sorting-isolated neurons. Both perturbation strategies produced the expected directional changes in target transcripts. Overexpression of either gene increased the other, whereas knockdown did not elicit reciprocal suppression, indicating asymmetric regulatory coupling. Phenotypically, Srebf2 showed a bidirectional association with senescence-like changes, as both overexpression and knockdown increased p16 and p21, whereas Zmiz1 acted more directionally, with overexpression increasing and knockdown reducing these markers. Transcriptomic profiling revealed broad direction-dependent remodeling, including a set of 55 genes that changed concordantly across perturbation directions. Pathway analysis further showed specialization, with Zmiz1 preferentially associated with an Alzheimer’s disease-related signature and Srebf2 more strongly linked to cholinergic synapse programs. Together, these findings identify Srebf2 and Zmiz1 as coupled but non-equivalent regulators of a neuronal senescence-like program.