Killing of single neurones by intracellular injection of proteolytic enzymes

Abstract

To approach problems of sprouting, regeneration, and specificity of neuronal connections, it would be useful if a technique were available for selectively destroying an individual neurone or group of neurones without damaging other cells. One established method for eliminating a class of cells is to use the genetic approach and produce mutants. This, however, is often impractical for animals with long generation times. In invertebrate preparations, surgical removal of the cell body is not sufficient, since neuronal processes can survive and continue to function normally for weeks or months after they have been severed from their cell bodies^1,2. We have used the central nervous system of the leech to establish a technique for destroying individual neurones one at a time. The leech is a convenient preparation since the nerve cells are large, easily recognisable, and many have been identified and their synaptic connections and peripheral fields are well known^3,4. We have found that Pronase, a mixture of proteolytic enzymes (Calbiochem Pronase, free of nucleases, B grade), can be injected by pressure into an identified cell. With time, it spreads throughout the neurone's central and peripheral processes and destroys them.