Analysis of axonal tracts and terminals of Brn3a-persistent spinal dorsal horn neurons.

(A) AAV carrying FLEX-switched tdTomato and SypEGFP genes was injected into the lumbar spinal cord (~L5) of Brn3aCre/+ mice. This experiment enables labeling of Brn3a-persistent neurons and their presynaptic terminals by tdTomato and SypEGFP, respectively. (B-M) Three weeks after AAV injection, the spinal cord and whole brain of the mice were dissected out to analyze the distribution of axonal tracts and terminals of Brn3a-persistent neurons. tdTomato-positive cells were distributed in the lumbar spinal dorsal horn on the right. (B) Dorsal view of the spinal cord of AAV-injected mice. Solid and broken lines indicate the outline and the center of the spinal cord, respectively. (C) A transverse section of the lumbar spinal cord. A solid line indicates the outline of the spinal cord whereas a broken line indicates the boundary between the gray and white matters. Scale, 500 μm. (D) Distribution of tdTomato (left) and SypEGFP (right) fluorescence throughout the spinal cord of AAV-injected mouse. The area where tdTomato-positive cells were distributed was about 1.5 mm along the rostrocaudal axis. Fluorescence image of the representative transverse section in this area is shown in “injection site”. Fluorescence images rostrally (+2.0 cm, +1.5 cm, +1.0 cm, and +0.5 cm) and caudally (-0.5 cm and -1.0 cm) away from the injection site are also shown. The axonal tract labeled with tdTomato is distributed in the ipsilateral dorsal funiculus (DF), ipsilateral dorsolateral funiculus (DLF), ipsilateral ventrolateral funiculus (VLF), contralateral ventral funiculus (VF), contralateral VLF, or ipsilateral deep dorsal horn (circled by dotted magenta line). The fluorescence of tdTomato and SypEGFP is pseudocolored in black. The axonal tract on the contralateral side was mainly distributed in the VF near the injection site (+0.5 cm), but was found in the VLF on the section distant from the injection site (+2.0 cm, +1.5 cm, and +1.0 cm). Throughout the spinal cord, accumulation of presynaptic terminal of Brn3a-persistent neurons labeled with SypEGFP was found in the ipsilateral deep dorsal horn 0.5 cm and 1.0 cm rostral to the injection site (dotted green line). This region roughly corresponds to the Clarke nucleus in the thoracic spinal cord. Solid red lines indicate the outline of the spinal cord, whereas dotted red lines indicate the boundary between gray and white matters. Data are representative of images from 2 mice. (E-M) The distribution of presynaptic terminals of Brn3a-persistent spinal dorsal horn neurons in the medulla (E-I) and pons (J-M). (E) Schematic of the medulla with the location of nuclei (dotted red line) are shown. GN, gracile nucleus; CuN, cuneate nucleus; NTS, nucleus of the solitary tract; LRN, lateral reticular nucleus; IO, inferior olivary nucleus. Fluorescence images of SypEGFP (marked in E) around the dorsal (F, G) and ventral (H, I) medulla are shown. Strong EGFP signal was found in the ipsilateral GN, whereas weaker signal was also seen in the contralateral GN, and NTS, LRN, and IO on both sides. Solid and dotted red lines indicate the outline of the brain and the boundary of nuclei, respectively. (J) Schematic of the pons with the location of nuclei (dotted red line) is shown. Fluorescence images of SypEGFP (marked in J) around the dorsal (K, L) and ventral (M) pons are shown. EGFP signal was seen in the lateral parabrachial nucleus on both sides (arrows) and ventral area of the pons on the ipsilateral side. Red dotted lines indicate the outline of neuronal tracts and nuclei. scp, superior cerebellar peduncle; vsc, ventral spinocerebellar tract; un, uncinate fasciculus of the cerebellum; 7n, facial nerve; rs, rubrospinal tract; DPO, dorsal periolivary nucleus; LSO, lateral superior olive. Scale in F-I and K-M, 200 μm. Data are representative of images from 2 mice. (PDF)