Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP⁺ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP⁺ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Parvalbumin (PV) neurons in the basal forebrain (BF) orchestrate cognitive functions via extensive brain-wide projections. However, the age-related cognitive decline of their anatomical circuits remains poorly understood. Here, we employed viral tracing and fluorescence micro-optical sectioning tomography (fMOST) to reveal the vulnerability of the BF-PV circuits during aging. Quantitative whole-brain fluorescence intensity analysis revealed that BF-PV neurons projecting to the medial mammillary nucleus (MM) exhibited pronounced age-dependent neurodegeneration, characterized by 81.1% fiber loss and axonal swelling, while those innervating hippocampal CA1 showed a 70.3% reduction in fiber density. Optogenetic interventions demonstrated that selective activation of the BF^PV-MM circuit can ameliorate cognitive deficits in old mice, significantly improving the novel object recognition index and its change rate. In contrast, modulation of the BF^PV-CA1 circuit showed no significant effects. Moreover, with the whole-brain dataset, we reconstructed the morphology of individual neurons, revealing structural divergence between MM- and CA1-projecting PV neurons. Taken together, our results delineate the optogenetic-targeted activation of the BF^PV-MM circuit, which can ameliorate age-related cognitive decline and provide both theoretical and therapeutic foundations for targeting neurodegenerative disorders.