Individual neurons in the leech can regenerate severed axons to reconnect with particular neurons that are their normal synaptic targets. In many animal species injured adult neurons may at first glance seem to revert to their developmental state, but there can be important differences between adult repair and embryonic development, including differences in synaptic function. Taking advantage of identifiable cells and cell types in the leech, the proposed project addresses the questions: (1) is axon sprouting controlled by certain cells and cellular interactions, (2) are particular molecules such as leech """"""""laminin"""""""" and surface markers on mechanosensory neurons associated with directed growth and repair, (3) do microglia play a critical role in regeneration, (4) can protein synthesis and transport in short axon systems, particularly during regeneration, be studied by injection of synthetic mRNA's, (5) how does cell structure determine synaptic function and connectivity, and (6) do mechanisms that produce successful repair recapitulate those that guide embryonic development? The methodology will include electrophysiological recording and intracellular injection of markers for subsequent light and electron microscopic examination. Growing axons and migrating cells labelled with fluorescent markers will be tracked in living preparations with the aid of low-light video enhancement. Properties of pathways and surfaces of particular cells that bind cell-specific ligands including monoclonal antibodies will be examined with electron microscopy. Identified neurons and axons will be injected with mRNA synthesized in vitro that codes for cytoplasmic and integral membrane proteins. Single cells will be killed with intracellular injection of proteases or focally lesioned with an argon laser and fluorescent dyes. The morphology of certain cells and the complex patterns of normal and regenerated synaptic contacts between them will be reconstructed with a computer. Growth of individual cells will be studied during development and compared with regeneration. Understanding mechanisms for development and accurate regeneration in the leech central nervous system might suggest ways that nerve regeneration in higher animals including humans could be made more reliable and precise.
Showing the most recent 10 out of 16 publications
