Three-dimensional evaluation of the spinal local neural network revealed by the high-voltage electron microscopy: A double immunohistochemical study

Three-dimensional (3-D) analysis of anatomical ultrastructures is important in biological research. However, 3-D image analysis on exact serial sets of ultrathin sections from biological specimens is very difficult to achieve, and limited information can be obtained by 3-D reconstruction from these sections due to the small area that can be reconstructed. On the other hand, the highpenetration power of electrons by an ultra-high accelerating voltage enables thick sections of biological specimens to be examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D analysis of the central nervous system because considerably thick sections can be observed at the ultrastructure level. Here, we applied HVEM tomography assisted by light microscopy to a study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by double-labeling immunohistochemistry. This powerful methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D ultrastructural level.