Three-dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS.

At birth, the opossum, Monodelphis domestica, corresponds roughly to a 14-day-old mouse embryo. The aim of these experiments was to compare the distribution of monoaminergic neurons in the two preparations during development and to follow their regeneration after injury. Procedures that allowed antibody staining to be visible in transparent whole mounts of the entire central nervous system (CNS) were devised. Neurons throughout the brain and spinal cord were stained for tyrosine hydroxylase (TH) and for serotonin (5-HT). At birth, patterns of monoaminergic cells in opossum CNS resembled those found in 14-day mouse embryos and other eutherian mammals. By postnatal day 5, immunoreactive cell bodies were clustered in appropriate regions of the midbrain and hindbrain, and numerous axons were already present throughout the spinal cord. Differences found in the opossum were the earlier presence of TH neurons in the olfactory bulb and of 5-HT neuronal perikarya in the spinal cord. Most, if not all, monoaminergic neurons in opossum were already postmitotic at birth. To study regeneration, crushes were made in cervical cords in culture. By 5 days, 8% of all TH-labeled axons and 14% of serotonergic axons had grown beyond lesions. Distal segments of monoaminergic axons degenerated. In CNS preparations from opossums older than 11 days, no regeneration of monoaminergic fibers occurred. Isolated embryonic mouse CNS also showed regeneration across spinal cord lesions, providing the possibility of using knockout and transgenic animals. Our procedures for whole-mount observation of identified cell bodies and their axons obviates the need for serial reconstructions and allows direct comparison of events occurring during development and regeneration.