ZFIN Figure: Nakamura et al., 2023, Figure 6

Figure 6

Cerebrospinal fluid-contacting neuron (CSF-cN) connections with premotor and motor neurons in the spinal cord.

(A) Diagram of Chat-GFP⁺ cholinergic neurons and adeno-associated virus (AAV)-Syn-mCherry labeled CSF-cNs in Chat^Cre;CAG-lcl-EGFP mice. Cc, central canal; CCC, central canal cluster cells; MN, motor neuron. Image positions of (D–K) are indicated in the right panel. (B, C) Representative images of mCherry⁺ CSF-cNs (red) and Chat-GFP⁺ neurons (green) in the cervical (B) and lumbar cord (C) of Chat^Cre;CAG-lcl-EGFP mice. (D) A magnified image of Chat-GFP⁺ (green) and mCherry⁺ fibers (red), which are intermingled at the ventral midline and ventral funiculus of the cervical cord. The dotted line indicates the border between the gray and white matter. (E, F) Chat-GFP⁺ neurites (green) extend near the central canal (yellow dotted lines) (E), and those with gephyrin⁺ puncta (blue and white, arrowheads) are contacted by mCherry⁺ CSF-cN fibers (red) (F). (F) is a magnified view of the white dotted area in (E). (G, H) Chat-GFP⁺ interneuron (green) near the central canal (yellow dotted lines) is contacted by mCherry⁺ CSF-cN fibers (red) apposing gephyrin⁺ puncta (blue and white, arrowheads). (H) is a magnified view of the white dotted area in (G). (I, J) Chat-GFP⁺ neurites intermingled with the mCherry⁺ subependymal axon bundle (red) in the cervical cord (arrowheads). Transverse (I) and horizontal sections (J). (K) Chat-GFP⁺ neurites (green) are intermingled with the mCherry⁺ axon bundle (red) in the ventral funiculus (arrowheads), with some making contact with the gephyrin⁺ puncta (blue and white, closed arrowheads). (L) Diagram representing axial MNs and CSF-cNs, labeled by retroAAV-CAG-EGFP and AAV-Syn-mCherry injections into the axial muscle and lateral ventricle, respectively. Image positions of (N, O) are indicated. (M) GFP⁺ MN of the dorsal neck muscle (semispinalis capitis) is labeled with retroAAV-CAG-EGFP (green) and mCherry⁺ CSF-cNs at the cervical level. Note the GFP⁺ fibers extending to the central canal, ventral midline, and ventral funiculus. (N) GFP⁺ MN neurites extending near the central canal and are contacted by mCherry⁺ CSF-cN fibers with GAD65⁺ puncta (blue and white, arrowheads). (O) GFP⁺ MN neurites (green) are intermingled with the mCherry⁺ axon bundle (red) in the ventral funiculus with GAD65⁺ puncta (blue, white, arrowheads). The right panel is a magnified view of the dotted area in the left panel. (P) Diagram of Chx10-GFP⁺ V2a neurons and AAV-Syn-mCherry-labeled CSF-cNs in Chx10^Cre;CAG-lcl-EGFP mice. Image positions of (R, S) are indicated. (Q) Chx10-GFP⁺ V2a neurons (green) and mCherry⁺ CSF-cNs (red) in the cervical cord of Chx10^Cre;CAG-lcl-EGFP mice. (R, S) Chx10-GFP⁺ fibers (green) extending to mCherry⁺ fiber bundles (red) in the ventral funiculus. (S) shows a magnified view of the dotted area in (R), which consists of Chx10-GFP⁺ fibers contacted by mCherry⁺ ventral bundles with GAD65⁺ puncta (blue and white) (arrowheads). (T) Serial block-face scanning electron microscopy images of the ventral bundles showing COX4-dAPEX2⁺ presynaptic terminals of CSF-cNs (pseudo-colored in green) in contact with a COX4-dAPEX2^– neurite coming from outside of the bundle (yellow). Positions 1 and 2 in the left panel are magnified in the right panels (the 80th and 139th serial images). (U–W) Transmission electron microscope images of presynaptic terminals containing COX4-dAPEX2⁺ mitochondria (arrowheads) contacting with COX4-dAPEX2^– neurites in the bundle of ventral funiculus at the thoracic level (U, V) and subependymal area at the cervical level (W). Scale bars, 250 µm (B, C, M, Q); 50 µm (D), (K), left panel of (O), (R); 20 µm (E), (G), (I), (J), (N), right panel of (O); 5 µm (F), (H), (S), left panel of (T); 0.5 µm (right panels of T, U, V, W).

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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