Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape.

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Songbird Ventral Pallidum Sends Diverse Performance Error Signals to Dopaminergic Midbrain.

Motor skills improve with practice, requiring outcomes to be evaluated against ever-changing performance benchmarks, yet it remains unclear how performance error signals are computed. Here, we show that the songbird ventral pallidum (VP) is required for song learning and sends diverse song timing and performance error signals to the ventral tegmental area (VTA). Viral tracing revealed inputs to VP from auditory and vocal motor thalamus, auditory and vocal motor cortex, and VTA. Our findings show that VP circuits, commonly associated with hedonic functions, signal performance error during motor sequence learning.

Vocal learning in songbirds requires cholinergic signaling in a motor cortex-like nucleus.

Cholinergic inputs to cortex modulate plasticity and sensory processing, yet little is known about their role in motor control. Here, we show that cholinergic signaling in a songbird vocal motor cortical area, the robust nucleus of the arcopallium (RA), is required for song learning. Reverse microdialysis of nicotinic and muscarinic receptor antagonists into RA in juvenile birds did not significantly affect syllable timing or acoustic structure during vocal babbling. However, chronic blockade over weeks reduced singing quantity and impaired learning, resulting in an impoverished song with excess variability, abnormal acoustic features, and reduced similarity to tutor song. The demonstration that cholinergic signaling in a motor cortical area is required for song learning motivates the songbird as a tractable model system to identify roles of the basal forebrain cholinergic system in motor control. NEW & NOTEWORTHY Cholinergic inputs to cortex are evolutionarily conserved and implicated in sensory processing and synaptic plasticity. However, functions of cholinergic signals in motor areas are understudied and poorly understood. Here, we show that cholinergic signaling in a songbird vocal motor cortical area is not required for normal vocal variability during babbling but is essential for developmental song learning. Cholinergic modulation of motor cortex is thus required for learning but not for the ability to sing.