Dorsal Horn of Mouse Lumbar Spinal Cord Imaged with CLARITY

Abstract

The organization of the mouse spinal dorsal horn has been delineated in 2D for the six Rexed laminae in our publication Atlas of the Spinal Cord: Mouse, Rat, Rhesus, Marmoset, and Human. In the present study, the tissue clearing technique CLARITY was used to observe the cyto- and chemoarchitecture of the mouse spinal cord in 3D, using a variety of immunohistochemical markers. We confirm prior observations regarding the location of glycine and serotonin immunoreactivities. Novel observations include the demonstration of numerous calcitonin gene-related peptide (CGRP) perikarya, as well as CGRP fibers and terminals in all laminae of the dorsal horn. We also observed sparse choline acetyltransferase (ChAT) immunoreactivity in small perikarya and fibers and terminals in all dorsal horn laminae, while gamma aminobutyric acid (GABA) and glutamate decarboxylase-67 (GAD67) immunoreactivities were found only in small perikarya and fibers. Finally, numerous serotonergic fibers were observed in all laminae of the dorsal horn. In conclusion, CLARITY confirmed the 2D immunohistochemical properties of the spinal cord. Furthermore, we observed novel anatomical characteristics of the spinal cord and demonstrated that CLARITY can be used on spinal cord tissue to examine many proteins of interest.

Figure 1

Cr (segment L1), GABA (segment L2), serotonin (segment L2), and GAD67 (segment L3) immunoreactive neurons and fibers in the mouse spinal dorsal horn. Scale bar is 100 μm. Med: medial; Lat: lateral.

Figure 2

GlyT2 (segment L2), NOS (segment L2), ChAT (segment L5), and CGRP (segment L2) immunoreactive perikarya (arrowheads) and fibers in the superficial laminae of the mouse spinal dorsal horn. Scale bar: 100 μm for GlyT2 and 200 μm for NOS, CHAT, and CGRP. Med: medial; Lat: lateral.