single neuron imaging 2D morphology 3D morphology VNC imaging tracing & identification mapping & visualization

Abdominal hemisegments in the embryo and larva comprise 30 highly stereotyped body wall muscles, each of which is innervated by one or more of an estimated 38 unique motor neurons. These motor neurons can be subdivided into distinct classes based on their association with specific nerve branches, and these subclasses can be further refined by morphological distinctions.

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The dendritic morphologies of individual motor neurons are typically difficult to resolve because of the vast number of different cell types in the larval CNS. To examine the dendritic arborization patterns of motor neurons in the larval CNS in detail, we use the mosaic analysis with a repressible cell marker (MARCM) system to genetically label single motor neuron clones with a membrane-targeted GFP (mCD8::GFP) [Kim et. al, Dev. Biol. 2009].

imaging single neurons

MARCM provides unparalleled single-cell resolution and further allows for the labeling of single motor neurons that are not easily accessible by dye-backfilling methods. To enhance imaging resolution, larvae with motor neuron clones are dissected to expose the CNS, fixed, and immuno-processed with anti-mCD8 (green channel) and anti-Fasciclin II (FasII) (red channel) antibodies. FasII labels axon fascicles that divide the central neuropil into distinct territories [Landgraf et. al, J. Neurosci 1997], providing a frame of reference in which to map the dorsal-ventral and mediolateral positions of the motor neuron soma and dendrites. The single motor neuron clones are then visually identified and the image stacks are acquired with confocal microscopy.