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Pipeline for fluorescent imaging and volumetric analysis of neurons in cleared mouse spinal cords.

ABSTRACT: Precisely measuring the number and somatic volume of neurons in the central nervous system at single-cell resolution is technically challenging. Here, we combine multiple techniques to address this challenge in optically cleared mouse spinal cords. We describe in vivo neuron labeling approaches, tissue-clearing technology, light sheet fluorescence microscopy, and machine learning-guided imaging analysis. This combination provides a precise determination of the cell number and somatic volume of any neuron population in the spinal cords.

SUBMITTER: Yu H

PROVIDER: S-EPMC9576748 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Pipeline for fluorescent imaging and volumetric analysis of neurons in cleared mouse spinal cords.

Yu Hao H Li Qiang Q Sandoval Alfredo A Gibbs Holly C HC English Amber A Dunn Tiffany T Moth John J Elahi Hajira H Chen Bo B

STAR protocols 20221012 4

Precisely measuring the number and somatic volume of neurons in the central nervous system at single-cell resolution is technically challenging. Here, we combine multiple techniques to address this challenge in optically cleared mouse spinal cords. We describe in vivo neuron labeling approaches, tissue-clearing technology, light sheet fluorescence microscopy, and machine learning-guided imaging analysis. This combination provides a precise determination of the cell number and somatic volume of a ...[more]

PMID: 36227743

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Expression data from embryonic mouse cervical spinal cords

Project description:Mammalian motor circuits control voluntary movements by transmitting signals from the central nervous system (CNS) to muscle targets. To form these circuits, motor neurons (MNs) must extend their axons out of the CNS. Although motor axon exit from the CNS is an indispensable phase of motor axon pathfinding, the underlying molecular mechanisms remain obscure. Here, we present the first identification of a genetic pathway that regulates motor axon exit from the vertebrate spinal cord, utilizing spinal accessory motor neurons (SACMN) as a model system. SACMN are a homogeneous population of spinal MNs whose axons leave the CNS through a discrete lateral exit point (LEP) and can be visualized by the expression of the cell surface protein, BEN. We show that the homeodomain transcription factor, Nkx2.9, is selectively required for SACMN axon exit and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9; loss of Nkx2.9 leads to a reduction in Robo2 mRNA and protein within SACMN and SACMN axons fail to exit the spinal cord in Robo2-deficient mice. Consistent with short-range interactions between Robo2 and Slit ligands regulating SACMN axon exit, Robo2-expressing SACMN axons normally navigate through LEP-associated Slits as they emerge from the spinal cord, and fail to exit in Slit-deficient mice. Our studies support the view that Nkx2.9 controls SACMN axon exit from the mammalian spinal cord by regulating Robo-Slit signaling.

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